Synthetic antigen constructs against Campylobacter jejuni

ABSTRACT

The invention relates to immunogenic synthetic constructs capable of inducing an immune response against  Campylobacter jejuni  ( C. jejuni ) in a subject comprising one or more monosaccharides comprising one or more MeOPN moieties. Specifically, the invention relates to immunogenic synthetic constructs capable of inducing an immune response against  Campylobacter jejuni  ( C. jejuni ) in a subject comprising one or more MeOPN→6 Gal monosaccharides. The invention also relates to compositions comprising the immunogenic synthetic constructs and methods of inducing an immune response against  C. jejuni  in a subject comprising administering the immunogenic synthetic constructs, and/or compositions comprising the immunogenic synthetic construct, to the subject.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional PatentApplication No. 62/075,399 filed Nov. 5, 2014, and the benefit of U.S.Provisional Patent Application No. 62/127,935 filed Mar. 4, 2015, thedisclosures of which are incorporated by reference herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 11, 2016 isnamed “103281nonprov_ST25.txt” and is 3.99 kilobytes in size.

FIELD OF THE INVENTION

The inventive subject matter of the instant invention relates toimmunogenic synthetic constructs capable of inducing an immune responseagainst Campylobacter jejuni (C. jejuni) in a subject. The inventivesubject matter of the instant invention also relates to compositionscomprising the immunogenic synthetic constructs as well as methods ofinducing an immune response against C. jejuni in a subject.

BACKGROUND OF THE INVENTION

Diarrheal diseases are a major cause of morbidity and mortality in thedeveloping world. Among the most frequent bacterial causes of diarrheaare enterotoxigenic Escherichia coli (ETEC), Shigella species, and C.jejuni. Indeed, C. jejuni is estimated to cause 2.5 million cases ofgastroenteritis annually in the United States and greater than 400million cases worldwide. In developing countries, C. jejunigastroenteritis is primarily a pediatric disease. The symptoms of C.jejuni gastroenteritis include diarrhea, abdominal pain, fever andsometimes vomiting. Stools usually contain mucus, fecal leukocytes andblood, although watery diarrhea is also observed. The disease iszoonotic, and wild and domesticated birds represent a major reservoir.C. jejuni is a major foodborne infection, most often being associatedwith contaminated poultry, but major outbreaks have been associated withwater or raw milk contamination.

In addition to causing gastroenteritis, C. jejuni can also cause severalundesirable post-infectious conditions, including inflammatory bowelsyndrome, and a spondyloarthropathy known as Reiter's Syndrome.Moreover, recent studies have indicated an association between C. jejuniinfections and malnutrition and growth stunting in young children inresource-limited settings.

Another possible debilitating complication of C. jejuni infection is thedevelopment of Guillain-Barré Syndrome (GBS), a post-infectiouspolyneuropathy that can result in paralysis (Allos, B. M., J. Infect.Dis 176 (Suppl 2):S125-128 (1997).) C. jejuni is one of a limited numberof bacteria that can endogenously synthesize sialic acid, a nine carbonsugar that is found in mammalian cells. The association between C.jejuni and GBS is reportedly due to molecular mimicry between the sialicacid containing-outer core of the lipooligosaccharide (LOS) present inC. jejuni and human gangliosides (Moran, et al., J. Endotox. Res. 3: 521(1996).) It is believed that antibodies generated by a human subjectagainst the LOS cores of C. jejuni may cause an undesirable autoimmuneresponse to neural tissue in the subject. Indeed, studies suggest thatLOS synthesis in Campylobacter is controlled by a number of genes,including genes encoding enzymes involved in the biosynthesis of sialicacid. The sialic acid is then incorporated into LOS. This is consistentwith the observed molecular mimicry of LOS and human gangliosides inGBS. (Aspinall, et al., Eur. J. Biochem., 213: 1029 (1993); Aspinall, etal., Infect. Immun. 62: 2122-2125 (1994); Aspinall, et al., Biochem 33:241 (1994); Salloway et al., Infect. Immun., 64: 2945 (1996).)

C. jejuni is a Gram-negative bacterium, having surface capsularpolysaccharides (CPSs) that are involved in colonization and invasionand against which serum antibodies are generated. Recent analysis of theCampylobacter genome sequence has resulted in the identification of acomplete set of capsule transport genes similar to those seen in typeII/III capsule loci in the Enterobactericeae (Parkhill et al., Nature,403: 665 (2000); Karlyshev et al., Mol. Microbiol., 35: 529 (2000).)Subsequent genetic studies in which site-specific mutations were made inseveral capsule transport genes indicate that the capsule is the majorserodeterminant of the Penner serotyping scheme (Karlyshev et al., Mol.Microbiol., 35: 529 (2000).) The Penner scheme is one of two majorserotyping schemes of campylobacters and was originally thought to bebased on lipopolysaccharide O side chains (Moran and Penner, J. Appl.Microbiol., 86:361 (1999).) It is now believed that the structurespreviously described as O side chains are, in fact, polysaccharidecapsules. Interestingly, although C. jejuni capsular moieties areimportant in serodetermination, and despite over 47 Penner serotypes ofC. jejuni having been identified, it is believed that most Campylobacterdiarrheal disease is caused by only a limited number of these serotypes.Therefore, only selected strains of C. jejuni, predicated onepidemiological studies, may provide suitable candidate strains fordevelopment of potential vaccine compositions.

Several immunogenic CPS-CRM₁₉₇ conjugates associated with prevalent C.jejuni serotypes have been created. (Monteiro et al., (2009) Infect.Immun. 77, 1128-1136; Bertolo, L, et al. (2012) Carbohy Res 366:45-49.)An immunogenic C. jejuni CPS conjugate vaccine capable of protectingnon-human primates against C. jejuni diarrhea has been developed.(Monteiro et al., (2009) Infect. Immun. 77, 1128-1136, U.S. Pat. No.9,084,809.) U.S. Pat. No. 9,084,809 describes, inter alia, an anti-C.jejuni immunogenic composition composed of a capsule polysaccharidepolymer of C. jejuni strain 81-176 (also referred to herein as serotypeHS23/36) that is capable of inducing an immune response in BALB/c mice.This reference teaches that the HS23/36 capsule polysaccharide comprisestrisaccharides of galactose, 3-O-methyl-6-deoxy-altro-heptose andN-acetyl glucosamine; specifically, the immunogenic polysaccharidepolymer comprises a repeating trisaccharide structure having the formula[→3)-α-D-Gal-(1→2)-6d-3-O-Me-α-D-altro-Hep-(1→3)-β-D-GlcNAc-(1→]containing an O-methyl-phosphoramidate at the O-2 position of Gal.Notwithstanding the promise of prototype vaccines, and despite theimportance of this organism to human disease, there are yet no licensed,commercially available vaccines against C. jejuni. Thus, there currentlyremains a need for improved immunogenic compositions and methods forpreventing or ameliorating diseases associated with C. jejuni infection.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to an immunogenicsynthetic construct capable of inducing an immune response againstCampylobacter jejuni (C. jejuni) in a subject, wherein said immunogenicsynthetic construct comprises one or more monosaccharides comprising oneor more MeOPN moieties. In a particular embodiment, the immunogenicsynthetic constructs comprise one or more MeOPN→6 Gal monosaccharides.

In yet another aspect, the invention relates to compositions comprisingan immunogenic synthetic construct capable of inducing an immuneresponse against C. jejuni in a subject, wherein said immunogenicsynthetic construct comprises one or more monosaccharides comprising oneor more MeOPN moieties. In a particular embodiment, the immunogenicsynthetic construct comprises one or more MeOPN→6 Gal monosaccharides.

In a further aspect, the invention relates to methods of inducing animmune response against C. jejuni in a subject comprising administeringto the subject an effective amount of an immunogenic syntheticconstruct, wherein said immunogenic synthetic construct comprises one ormore monosaccharides comprising one or more MeOPN moieties. In aparticular embodiment, the immunogenic synthetic construct comprises oneor more MeOPN→6 Gal monosaccharides. In a particular embodiment, themethods may further comprise administering one or more boosting doses ofthe immunogenic synthetic construct. In particular embodiments, theeffective amount is an amount from about 0.1 μg to about 10 mg ofimmunogenic synthetic construct.

In a further aspect, the invention relates to methods of inducing animmune response against C. jejuni in a subject comprising administeringto the subject an effective amount of a composition comprising animmunogenic synthetic construct, wherein the immunogenic syntheticconstruct comprises one or more monosaccharides comprising one or moreMeOPN moieties. In a particular embodiment, the immunogenic syntheticconstruct comprises one or more MeOPN→6 Gal monosaccharides. In aparticular embodiment, the methods may further comprise administeringone or more boosting doses of the immunogenic synthetic construct. Inparticular embodiments, the effective amount is an amount from about 0.1μg to about 10 mg of immunogenic synthetic construct.

In various additional aspects, the invention relates to an immunogenicsynthetic construct for use in inducing an immune response against C.jejuni in a subject, wherein said immunogenic synthetic constructcomprises one or more monosaccharides comprising one or more MeOPNmoieties. In a particular embodiment, the immunogenic syntheticconstruct comprises one or more MeOPN→6 Gal monosaccharides. In anotheraspect, the invention relates to use of an immunogenic syntheticconstruct for inducing an immune response against C. jejuni in a subjectwherein said immunogenic synthetic construct comprises one or moremonosaccharides comprising one or more MeOPN moieties. In a particularembodiment, the immunogenic synthetic construct comprises one or moreMeOPN→6 Gal monosaccharides. In another aspect, the invention relates touse of an immunogenic synthetic construct in the manufacture of amedicament for inducing an immune response against C. jejuni in asubject, wherein said immunogenic synthetic construct comprises one ormore monosaccharides comprising one or more MeOPN moieties. In aparticular embodiment, the immunogenic synthetic construct comprises oneor more MeOPN→6 Gal monosaccharides.

In an additional aspect, the invention relates to a compositioncomprising an immunogenic synthetic construct for use in inducing animmune response against C. jejuni in a subject, wherein the immunogenicsynthetic construct comprises one or more monosaccharides comprising oneor more MeOPN moieties. In a particular embodiment, the immunogenicsynthetic construct comprises one or more MeOPN→6 Gal monosaccharides.In another aspect, the invention relates to use of a compositioncomprising an immunogenic synthetic construct for inducing an immuneresponse against C. jejuni in a subject, wherein the immunogenicsynthetic construct comprises one or more monosaccharides comprising oneor more MeOPN moieties. In a particular embodiment, the immunogenicsynthetic construct comprises one or more MeOPN→6 Gal monosaccharides.In another aspect, the invention relates to use of a compositioncomprising an immunogenic synthetic construct in the manufacture of amedicament for inducing an immune response against C. jejuni in asubject, wherein the immunogenic synthetic construct comprises one ormore monosaccharides comprising one or more MeOPN moieties. In aparticular embodiment, the immunogenic synthetic construct comprises oneor more MeOPN→6 Gal monosaccharides.

In an additional aspect, the invention relates to a pharmaceuticalcomposition comprising an immunogenic synthetic construct for use ininducing an immune response against C. jejuni in a subject, wherein theimmunogenic synthetic construct comprises one or more monosaccharidescomprising one or more MeOPN moieties. In a particular embodiment, theimmunogenic synthetic construct comprises one or more MeOPN→6 Galmonosaccharides. In another aspect, the invention relates to use of apharmaceutical composition comprising an immunogenic synthetic constructfor inducing an immune response against C. jejuni in a subject, whereinthe immunogenic synthetic construct comprises one or moremonosaccharides comprising one or more MeOPN moieties. In a particularembodiment, the immunogenic synthetic construct comprises one or moreMeOPN→6 Gal monosaccharides. In another aspect, the invention relates touse of a pharmaceutical composition comprising an immunogenic syntheticconstruct in the manufacture of a medicament for inducing an immuneresponse against C. jejuni in a subject, wherein the immunogenicsynthetic construct comprises one or more monosaccharides comprising oneor more MeOPN moieties. In a particular embodiment, the immunogenicsynthetic construct comprises one or more MeOPN→6 Gal monosaccharides.

In an additional aspect, the present invention is directed to methods ofsynthesizing the immunogenic synthetic constructs of the instantinvention.

In various embodiments of the aforementioned aspects, the immunogenicsynthetic construct may be conjugated to a carrier compound, e.g., acarrier protein. In a particular embodiment, the carrier proteincontains at least one T-cell epitope. In a particular embodiment, thecarrier protein is CRM₁₉₇.

In additional embodiments of the aforementioned aspects, the compositionis a pharmaceutical composition. In a particular embodiment, thepharmaceutical composition is a vaccine formulation.

In particular embodiments, the pharmaceutical compositions and thevaccine formulations may comprise one or more adjuvants. In particularembodiments, the adjuvant is selected from the group consisting oftoll-like receptor ligands, aluminum phosphate, aluminum hydroxide,monophosphoryl lipid A, liposomes, and derivatives and combinationsthereof. In further embodiments, the pharmaceutical compositions andvaccine formulations comprise one or more additional immunoregulatoryagents. In a particular embodiment, the immunoregulatory agent is asubstance selected from the group consisting of antigens of one or morestrains of C. jejuni, antigens of ETEC, Shigella lipopolysaccharidestructures, and unconjugated carrier proteins.

In particular embodiments, the methods of inducing an immune responseagainst C. jejuni in a subject comprise administering the constructconjugated to a protein carrier. In a particular embodiment, the proteincarrier is CRM₁₉₇. In another particular embodiment, the method furthercomprises administering the construct or conjugate with one or moreadjuvants. In a particular embodiment, the adjuvant is selected from thegroup consisting of toll-like receptor ligands, aluminum phosphate,aluminum hydroxide, monophosphoryl lipid A, liposomes, and derivativesand combinations thereof. In particular embodiments of theaforementioned aspects, the subject is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the CPS repeating blocks of serotype complexes HS1, HS3,HS4, and HS23/36 and the strain specific heptose units and O-methylphosphoramidate (MeOPN) linkages. Abbreviations: “±”, MeOPN moieties innon-stoichiometric amounts; Gal, galactose; Gro, glycerol; Fru,fructose; Hep, heptose; GlcpNAc, N-acetyl-D-glucosamine. The existenceof MeOPN-6-Gal in strain HS23/36 is based on the discovery reportedherein.

FIG. 2 depicts synthesis of the p-methoxyphenyl glycoside of theMeOPN→6-Gal construct (O-Me-phosphoramidate galactoside),MeOPN→6-α-D-Galp-(1→OMP (“Scheme 1”.) The reagents and conditionsemployed in the steps indicated therein are as follows: (a) TrCl,pyridine, 95%; (b) AlIBr, NaH, DMF, 0° C., 89%; (c) 80% AcOH, 80° C.,78%; (d) PCl₂(O)OMe, Et₃N, CH₂Cl₂, then NH₃(g), 19%; (e) PdCl₂, MeOH,39%. Tr, trityl; All, allyl; DMF, dimethyl formamide; OMP,4-methoxyphenyl group.

FIG. 3 depicts synthesis of the aminopentyl glycoside of the MeOPN→6-Galconstruct (O-Me-phosphoramidate galactoside),MeOPN→6-β-D-Galp-(1→O(CH₂)₅NH₂ (“Scheme 2”.) The reagents and conditionsemployed in the steps indicated therein are as follows: (a) CAN, CH₃CN,H₂O, 0° C.; then CCl₃CN, K₂CO₃, CH₂Cl₂, 57% over 2 steps; (b)HO(CH₂)₅NPhth, TMSOTf, CH₂Cl₂, 65%; (c) 80% AcOH, 80° C., 78%; (d)PCl₂(O)OMe, Et₃N, CH₂Cl₂, then NH_(3(g)), 27%; (e) PdCl₂, MeOH, 75%, (f)H₂NNH₂, EtOH, 82%. CAN, cerium ammonium nitrate; TMSOTf, trimethylsilyltrifluoromethanesulfonate; Tr, trityl; All, allyl; OMP, 4-methoxyphenylgroup; OTCA, trichloroacetimidate.

FIG. 4 depicts another scheme for the synthesis of the aminopentylglycoside of the MeOPN→6-Gal construct (O-Me-phosphoramidategalactoside), MeOPN→6-β-D-Galp-(1→O(CH₂)₅NH₂ (“Scheme 2a”.) The reagentsand conditions employed in the steps indicated therein are as follows:(a) TrCl, pyridine, 95%; (b) AlIBr, NaH, DMF, 0° C., 89%; (c) CAN,CH₃CN, H₂O, 0° C.; then CCl₃CN, K₂CO₃, CH₂Cl₂, 57% over 2 steps; (d)HO(CH₂)₅NPhth, TMSOTf, CH₂Cl₂, 65%; (e) 80% AcOH, 80° C., 78%; (f)PCl₂O₂Me₂, Et₃N, CH₂Cl₂, then NH₃(g), 27%; (g) PdCl₂, MeOH, 75%, (h)H₂NNH₂, EtOH, 82%. CAN, cerium ammonium nitrate; TMSOTf, trimethylsilyltrifluoromethanesulfonate; Tr, trityl; All, allyl; OMP, 4-methoxyphenylgroup; OTCA, trichloroacetimidate.

FIG. 5 depicts the synthesis of MeOPN→2-β-D-Galp-(1→OMP (“Scheme 3”.)The reagents and conditions employed in the steps indicated therein areas follows: (a) AlIBr, NaH, DMF, 0° C., 95%; (b) 80% AcOH, 80° C., 94%;(c) BzCl, pyridine, 97%; (d) PdCl₂, MeOH, 92%; (e) PCl₂(O)OMe, Et₃N,CH₂Cl₂, then NH₃(g), 26%; (f) NaOMe, MeOH, 73%. All, allyl; Bz, Benzoyl.

FIG. 6 depicts location of possible MeOPN moieties and capsulecross-reactivity to MeOPN-6-Gal with antibodies to multiple conjugatevaccines. FIG. 6(A) depicts the structure of possible MeOPN modifiedmonosaccharides on MeOPN-6 Gal in the CPS of the HS 23/36 serotype of C.jejuni. All “R” groups present can stand for either H or MeOPN, i.e.,each site of modification (Gal-2 or Gal-6) can be substituted witheither H or MeOPN. FIG. 6 (B) depicts the structure of MeOPN modifiedmonosaccharide in the CPSs of the indicated serotypes of C. jejuni,HS:4, HS: 1, and HS:3. In order to test for capsule cross-reactivity, aspot of MeOPN-6-Gal was combined with the indicated detectinganti-CRM₁₉₇ conjugate antiserum (indicated on the right side of theblot). Data indicate that antibodies to HS23/36, HS4 and HS1 serotypesof C. jejuni can react with the synthetic MeOPN-6-Gal construct.

FIG. 7 depicts the immunodetection of MeOPN→6-α-D-Galp-(1→OMP (column A)and MeOPN→6-β-D-Galp-(1→O—(CH₂)₅NH₂ (column B) by C. jejuni CPSconjugate antisera of serotypes HS1 (1:500), HS3 (1:500), HS4 (1:2000)and HS23/36 (1:2000) as indicated in the center column. Dilutions weredone in TBST (20 mM Tris, pH7.4, 0.425 N NaCl, 0.05% Tween 20.) Datashow that antibodies to HS23/36, HS4 and HS1 serotypes of C. jejuni canreact with the synthetic MeOPN-6-Gal construct either with or withoutadded linker.

FIG. 8 depicts an immunoblot which demonstrates that rabbit antibodiesto an HS23/36 polysaccharide-CRM₁₉₇ conjugate vaccine detectedMeOPN-6-Gal, but did not detect isomers of MeOPN-2-Gal. These dataclearly indicate the immunogenicity of the MeOPN-6-Gal monosaccharideand the immunodominance of the methyl phosphoramidate at the 6 positionof Gal over MeOPN at the 2 position of Gal.

FIG. 9 depicts the conjugation of the linker-equipped galactoside withcarrier protein, CRM₁₉₇ (CRM₁₉₇ is depicted as ribbon diagram) (“Scheme4”.) The reagents and conditions employed in the steps indicated thereinare as follows: (a) di-N-hydroxy-succinimidyl adipate ester, Et₃N DMSO;(b) CRM₁₉₇, 70 mM NaPi, pH 7.0.

FIG. 10 depicts the analysis and confirmation of conjugation of linkerequipped galactoside with carrier protein: (A) Gel electrophoresis ofCRM₁₉₇ and MeOPN→6-β-D-Gal CRM₁₉₇ (compound 14); (B) Western blot ofMeOPN→6-β-D-Gal CRM₁₉₇ (compound 14) with C. jejuni HS23/36 whole cellantisera; and (C) the MALDI-TOF/MS of MeOPN→6-β-D-Gal CRM₁₉₇ (compound14.) The MeOPN-6-Gal-CRM₁₉₇ vaccine gave a major peak of mass61,781.206. The mass for CRM₁₉₇ in a similar MALDI experiment was 57,967daltons (not shown.) Thus, the mass difference was about 3,814 daltons.Since the mass of MeOPN-6-Gal and the linker is 461 daltons (data notshown), this indicates that approximately 8 MeOPN-6-Gal-linker moietieswere added per CRM₁₉₇ molecule.

FIG. 11 depicts flow cytometry analysis of C. jejuni HS23/36 cells withantisera raised by HS23/36 CPS conjugate (peak between approx. 10³-10⁴)and synthetic MeOPN→6-β-D-Gal CRM₁₉₇ conjugate 14 (peak between approx.0 and −10³). Peak at 0 represents binding of secondary antibody alone.APC-A, Allophycocyanin. Data demonstrate that a synthetic conjugatevaccine of the invention is capable of conjuring up antibodies inrabbits specific to the CPS MeOPN→6-D-Gal linkage exposed on thecell-surface of C. jejuni HS23/36 cells.

FIG. 12 depicts a summary of the synthesis of the MeOPN-6-Galmonosaccharide construct and conjugation to the carrier protein CRM₁₉₇.Ac, acetyl; MP, Methoxyphenyl; All, allyl; Tr, trityl; Phth,phthalimido.

FIG. 13 depicts ³¹P NMR (A) and ¹H NMR (B) spectra ofMeOPN→6-α-D-Galp-(1→OMP performed using conventional methods.

FIG. 14 depicts depicts ³¹P NMR (A) and ¹H NMR (B) of 4-Methoxyphenyl2-O-methyl-phosphoramidyl-β-D-galactopyranoside performed usingconventional methods.

FIG. 15 depicts the synthesis of a synthetic polymeric conjugate of theinvention comprising multiple MeOPN-6-Gal monosaccharides chemicallyassociated using a starch backbone which is equipped with a linker andconjugated to the carrier protein, CRM₁₉₇.

FIG. 16 depicts a 12.5% SDS-PAGE gel (sodium dodecylsulfate-polyacrylamide gel electrophoresis) (A) and immunoblot (B) ofthe synthetic polymeric construct of FIG. 15 comprising multipleMeOPN-6-Gal monosaccharides. As indicated, FIG. 16(A) includes lanes forthe molecular weight marker, the synthetic construct, and carrierprotein alone. FIG. 16(B) provides the blot of the construct. The geland blot were prepared using conventional methods as described inExample 7.

FIG. 17 depicts ¹H NMR of the synthetic polymeric construct of FIG. 15showing the successful attachment of the C. jejuni MeOPN-6-Gal syntheticantigen to the modified (oxidized) starch polymer. X axis is ppm. Thearrow at approximately 4.5 ppm indicates the p-anomeric signal of 6MeOPN-β-D-Gal synthetic antigen; the remaining arrows indicate CH₂signals of the linker.

FIG. 18 depicts another synthetic polymeric construct of the inventioncomprising multiple MeOPN-6-Gal monosaccharides chemically associatedwith other saccharides using a starch backbone and which is equippedwith a linker and conjugated to a carrier protein. Specifically, asdepicted, the synthetic polymer comprises multiple MeOPN-6-Gal,MeOPN-2-Gal, and MeOPN-1-Fru monosaccharides.

FIG. 19 depicts the structure of two repeats of the 81-176 capsulartrisaccharide (A). The position of MeOPN-2-Gal and MeOPN-6-Gal isindicated. FIG. 19 (B) depicts a cartoon of genes in the variable CPSlocus of 81-176. The variable CPS locus of 81-176 maps between kpsC(CJJ81176_1413c) and kpsF (CJJ81176_1437c) shown in grey and encompasses22 genes. Genes of known function are labeled. Those genes that involvedin synthesis of MeOPN are labeled as mpnA-D ([21]) and the remaininggenes labeled are involved in heptose synthesis. Genes in blackrepresent the two putative MeOPN transferases, CJJ81176_1420 andCJJ81176_1435.

FIG. 20 depicts an immune response of anti-conjugate antibodies. A.Immunoblot with rabbit hyperimmune antiserum to formalin-killed wholecells of C. jejuni 81-176. B. Immunoblot with rabbit hyperimmuneantiserum to an 81-176-CRM197 conjugate vaccine (CJCV1). Lanes are:Marker, Precision Plus Protein Standards; wildtype 81-176; strain 3468,a kpsM, non-encapsulated mutant, ([34]); strain 3469, the complement ofkpsM, strain 3390, the mpnC mutant lacking the ability to synthesizeMeOPN ([21]); strain 3391, the complement of mpnC; strain 3477, themutant in CJJ81176_1420; strain 3498, the complement of theCJJ81176_1420 mutant; strain 3636, the mutant in CJJ81176_1435; strain3637, the complement of the CJJ81176_1435 mutant. C. Immune response ofanti-conjugate antibodies to purified CPS polysaccharide as measured byELISA. Strain numbers of the mutants used are indicated and explained inTable 1.

FIG. 21 depicts a 1D ³¹P NMR spectra showing the three distinctMeOPN-associated resonances (X, Y and Z) discussed in this work. A. CPSof C. jejuni 81-176 wild-type that contains only one MeOPN units (peakY). B. CPS of C. jejuni 81-176 wild-type that contains two MeOPN units(peak Y and Z). C. CPS of C. jejuni CJJ81176_1435 (3636) that contains anew MeOPN CPS modification (peak X).

FIG. 22 depicts a 1D slices from a 2D 1H-31P Heteronuclear Multiple BondCorrelation NMR experiment. A. CPS of C. jejuni 81-176 wild-type showingthe through bond correlation between MeOPN and 2-position of galactose.B. CPS of C. jejuni CJJ81176_1435 (3477) showing the through bondcorrelation between MeOPN and 6-position of galactose. C. CPS of C.jejuni CJJ81176_1420 (3636) showing the through bond correlation betweenMeOPN and an unidentified CPS position. HOD represents the position ofwater peak in each experiment.

FIG. 23 depicts the characterization of monoclonal DB3. A. Dot blot ofwhole cells of wildtype 81-176 and various mutants detected with DB3. B.Flow cytometry of wildtype, 3390, and 3391 with DB3. C. Flow cytometryof wildtype, 3636 and 3637 with DB3. D. Flow cytometry of wildtype,3477, and 3498 with DB3. The peak labeled “2°” in B, C and D showsbinding of the secondary antibody alone.

FIG. 24 depicts the variation of MeOPN levels of different batches ofconjugate vaccines. A. DB3 ELISA of three different batches of81-176-CRM197 conjugate vaccines. B. Endpoint titers of rabbitpolyclonal hyperimmune sera to capsules purified from wildtype 81-176(black bars) and the mpnC mutant (3390; gray bars). C-E, Flow cytometrycomparing binding of rabbit hyperimmune serum against conjugate CCV (C),DB4 (D) and CJCV1, (E) to wildtype 81-176, 3390, the mpnC mutant and3469, the kpsM mutant.

FIG. 25 depicts the resistance of C. jejuni strains to increasingamounts of NHS. Bacteria were exposed to increasing amounts of NHS for 1h at 37° C. and survivors enumerated by plate counts. Genotypes of thestrains are shown in Table 1. Strain 3636 was significantly differentfrom wildtype at all four concentration of NHS (P<0.05). Strain 3477 wassignificantly less serum resistant than wildtype at 5% NHS (P<0.05), 10%(P<0.005) and 15% (P<0.05). There was no significant difference in thecomplements of the two mutants, 3498 and 3637, with wildtype at anyconcentration of NHS. The double transferase mutant, 3479, wassignificantly lower than wildtype at 5% (P<0.0005), 10% (P<0.005), and15% NHS (P<0.05).

FIG. 26 depicts the phase variation of the CJJ81176_1435 MeOPNtransferase. A. Immunoblot of representative single colonies of 81-176with DB3. The intensity of reactivity is shown by the numerical scoring(3+, 2+, 1+ and negative). WT, the reaction of the population ofwildtype 81-176; the negative control is the population of the mpnCmutant, 3390, B. The percentage of colonies within the populationshowing differing levels of reactivity with DB3. The percentage ofcolonies that scored as 1+, 2+ and 3+ are shown; the area in blackrepresents colonies that were negative.

DETAILED DESCRIPTION

While the specification concludes with the claims particularly pointingout and distinctly claiming the invention, it is believed that thepresent invention will be better understood from the followingdescription.

All percentages and ratios used herein are by weight of the totalcomposition unless otherwise indicated herein. All temperatures are indegrees Celsius unless specified otherwise. All measurements made are at25° C. and normal pressure unless otherwise designated. The presentinvention can “comprise” (open ended) or “consist essentially of” thecomponents of the present invention as well as other ingredients orelements described herein. As used herein, “comprising” means theelements recited, or their equivalent in structure or function, plus anyother element or elements which are not recited. The terms “having” and“including” are also to be construed as open ended unless the contextsuggests otherwise. As used herein, “consisting essentially of” meansthat the invention may include ingredients in addition to those recitedin the claim, but only if the additional ingredients do not materiallyalter the basic and novel characteristics of the claimed invention.

All ranges recited herein include the endpoints, including those thatrecite a range “between” two values. Terms such as “about,” “generally,”“substantially,” and the like are to be construed as modifying a term orvalue such that it is not an absolute, but does not read on the priorart. Such terms will be defined by the circumstances and the terms thatthey modify as those terms are understood by those of skill in the art.This includes, at very least, the degree of expected experimental error,technique error and instrument error for a given technique used tomeasure a value. Unless otherwise indicated, as used herein, “a” and“an” include the plural, such that, e.g., “a MeOPN-6-Gal monosaccharide”can mean at least one MeOPN-6-Gal monosaccharide, as well as a pluralityof MeOPN-6-Gal monosaccharides, i.e., more than one MeOPN-6-Galmonosaccharide.

Where used herein, the term “and/or” when used in a list of two or moreitems means that any one of the listed characteristics can be present,or any combination of two or more of the listed characteristics can bepresent. For example, if a vaccine formulation against C. jejuni isdescribed as containing characteristics A, B, and/or C, the vaccineformulation against C. jejuni can contain A feature alone; B alone; Calone; A and B in combination; A and C in combination; B and C incombination; or A, B, and C in combination. The entire teachings of anypatents, patent applications or other publications referred to hereinare incorporated by reference herein as if fully set forth herein.

Until recently, MeOPN-2-Gal was thought to be the only MeOPN moiety onCPS Gal in C. jejuni strain 81-176 (otherwise referred to herein asserotype HS23/26.) (Kanipes et al., (2006) J Bacteriol. 188, 3273-3279.)By performing genetic and structural analyses of C. jejuni strainHS23/36, however, the inventors have surprisingly discovered a seconddistinct MeOPN at the O-6-position of the CPS Gal. As reported herein,the inventors have discovered that although present innon-stoichiometric amounts, CPS epitopes containing MeOPN units are keyC. jejuni immunogenic markers. Moreover, by performing comprehensiveimmunological analyses of multivalent conjugate vaccines using nativeCPSs of C. jejuni HS23/36, the inventors have discovered that theMeOPN-6-Gal monosaccharide is immunogenic and immunodominant overMeOPN-2-Gal and unmodified polysaccharide.

In view of the foregoing, the present invention is directed to animmunogenic synthetic construct capable of inducing an immune responseagainst C. jejuni in a subject. Specifically, in contrast to previousanti-C. jejuni immunogenic polysaccharide constructs or CPS conjugatevaccines, the instant invention is directed to an immunogenic syntheticconstruct against C. jejuni comprising one or more methyl phosphoramidylmonosaccharides, i.e., an immunogenic synthetic construct comprising oneor more O-methyl phosphoramidate (MeOPN) moieties, including but notlimited to, MeOPN at the 6 position of galactose.

In a particular embodiment, as specifically described in detail herein,the enhanced immunogenicity and efficacy of a synthetic MeOPN→6 Galconstruct against C. jejuni has surprisingly been discovered. Thus, invarious aspects, the invention includes a synthetic saccharide constructthat comprises one or more synthetic MeOPN→6 Gal monosaccharides,compositions comprising these synthetic saccharide constructs, andmethods of using these synthetic saccharide constructs. In a particularembodiment, the synthetic saccharide construct is conjugated to acarrier protein. Compositions, e.g., pharmaceutical anti-C. jejuniformulations, including vaccine formulations, comprising the syntheticconstruct (unconjugated or conjugated to a carrier protein) arecontemplated herein. Also contemplated herein are methods of inducing animmune response against C. jejuni in a subject comprising administeringto the subject an effective amount of the synthetic construct and/or acomposition of the instant invention, e.g., a vaccine formulation,comprising the synthetic construct in conjugated and/or unconjugatedforms.

The immunogenic synthetic constructs and conjugates of the instantinvention are believed to offer multiple advantages over previousconjugate vaccines made from purified C. jejuni capsule polysaccharides.For example, data indicate that MeOPN moieties are phase variable in C.jejuni, thus the level of this epitope normally present in vaccineformulations obtained from purified capsules can vary. As a result ofthis natural variability, different preparations from the same strain ofC. jejuni may have different levels of this MeOPN epitope, and thusdifferent immunogenicity. In contrast, by using a synthetic approach, apharmaceutical formulation (e.g., a vaccine formulation) comprising adesired level of MeOPN epitopes can be obtained, and provides theadvantage that the potential immunogenicity of the vaccine may becontrolled. In addition, as evident from the examples provided herein,the synthetic C. jejuni monosaccharide construct antigen of the instantinvention appears to have broader coverage than polysaccharides, thuspotentially reducing the valency required for a vaccine against C.jejuni. Thus, it is contemplated herein that the synthetic constructsdisclosed herein are antigenic determinants that can be used aseffective antigens in a vaccine formulation in which a single epitopecould cross-protect across more than one C. jejuni serotype. Moreover,since the use of the synthetic construct of the instant inventioneliminates the need to grow C. jejuni (a fastidious organism) and topurify the capsule, the synthetic construct is more cost-effective andthus provides a commercial advantage compared to other vaccines whichuse purified CPS.

In addition to the foregoing, the synthetic construct of the instantinvention is not only immunogenic, but also provides the advantage thatthe synthetic approach precludes concerns about development ofautoimmunity because the method does not require purification ofcapsules away from C. jejuni lipooligosaccharides (LOS) which oftencontains structures that mimic human gangliosides structurally and caninduce an autoimmune response that results in Guillain Barré Syndrome.

As understood by one of skill in the art, “MeOPN→6 Gal”, “MeOPN-6-Gal”,“MeOPN-6-Gal construct” and like terms refer to a galactosemonosaccharide which is modified to include an O-methyl phosphoramidatemoiety at the O-6 position of the galactose monosaccharide. Asunderstood herein, the synthetic MeOPN-6-Gal construct may comprisevarious other “R” groups in addition to the MeOPN moiety. The termencompasses constructs of various modified forms, e.g.,MeOPN→6-α-D-Galp-(1→OMP, i.e., 4-Methoxyphenyl6-O-methyl-phosphoramidate-α-D-galactopyranoside; as well as activatedforms including a linker, e.g., as MeOPN→6-β-D-Galp-(1→O(CH₂)₅NH₂, i.e.,5-Amino-pentanyl 6-O-methylphosphoramidate-β-D-galactopyranoside.Similarly, “MeOPN-2-Gal” and like terms refers to an O-methylphosphoramidate moiety at the O-2 position of the galactosemonosaccharide.

As understood herein, an “immunogenic synthetic construct” or moresimply “synthetic construct”, and like terms, refer to an in vitro,i.e., chemically produced, non-naturally occurring (“man-made”) compoundcomprising one or more monosaccharides comprising one or more MeOPNmoieties capable of inducing an immune response against Campylobacterjejuni (C. jejuni) in a subject. In a particular embodiment, theimmunogenic synthetic construct comprises one or more MeOPN→6 Galmonosaccharides which can elicit an immune response to C. jejuni in asubject. The MeOPN→6 Gal monosaccharide may also comprise various other“R” groups in addition to the MeOPN moiety. As contemplated herein, in aparticular embodiment, the immunogenic synthetic construct of theinstant invention comprises one or more synthetic MeOPN→6 Galmonosaccharides, alone or chemically associated in combination with oneor more other saccharides, and/or chemical linkers. For example, it iscontemplated herein that the synthetic construct of the presentinvention can comprise one or more additional monosaccharides incombination with one or more MeOPN-6-Gal monosaccharides.Monosaccharides found in the CPS of C. jejuni are particularlycontemplated herein, e.g., one or more of fructose, galactose, glucose,or heptose monosaccharides, and optionally substituted with one or moreadditional MeOPN moieties, including but not limited to, MeOPN-2-Gal, orother antigens against C. jejuni.

As discussed below in detail, it is contemplated herein that thesynthetic constructs of the instant invention, including syntheticconstructs comprising one or more MeOPN→6 Gal monosaccharides, may beactivated and conjugated to a carrier protein or may be used in anunconjugated form. When conjugated to a carrier protein, the syntheticconstruct may be referred to herein as a “conjugate vaccine” or as a“conjugate.”

As used herein, “a subject” includes an animal, including but notlimited to birds and mammals. Human beings are also encompassed in thisterm. As particularly contemplated herein, subjects include, e.g., anyanimal or human that has been infected with, or is at risk of beinginfected with, C. jejuni. A subject may be naïve, or non-naïve withregard to C. jejuni exposure. In particular, suitable subjects(patients) include, but are not limited to, farm animals (e.g.,chickens) as well as non-human primates and human patients.

As understood herein, the synthetic construct of the instant inventionmay be administered to a subject in order to induce an immune responsein the subject and thus prevent and/or ameliorate one or morepathological conditions associated with C. jejuni in the subject. Asunderstood herein, the concept of “inducing” an immune response in asubject refers to triggering a humoral and/or cellular immune responsein the subject.

The concept of “preventing and/or ameliorating” one or more pathologicalconditions associated with C. jejuni encompasses, e.g., averting orhindering the onset or development of a pathological conditionassociated with C. jejuni infection, as well as treating, curing,retarding, and/or reducing the severity of one or more pathologicalconditions associated with C. jejuni.

As used herein, the term “one or more pathological conditions associatedwith C. jejuni” refers to an undesirable condition in a subject causedby infection with C. jejuni (“campylobacteriosis”.) As contemplatedherein, such pathological conditions include clinical conditions anddiseases which may arise in a subject upon infection with C. jejuni, aswell as conditions which may develop in a subject as a consequence of aprevious instance of campylobacteriosis. These conditions are familiarto one of skill in the art and include, but are not limited, tocampylobacter gastroenteritis, Reiter's Syndrome, inflammatory bowelsyndrome, and Guillain-Barré Syndrome (GBS.)

Synthesis of the synthetic constructs of the instant invention,including the controlled synthesis and introduction of MeOPN to a simplesugar, activation of the resulting synthetic construct, addition of achemical linker, and conjugation of a carrier protein may be performedusing commercially available materials and methodologies familiar to acarbohydrate chemist. Particular methods of synthesis (synthesisschemes) are described in detail in the below examples. It iscontemplated herein that the methods of synthesizing the compoundsdisclosed in the below examples and “schemes” are included among theaspects of the instant invention.

As understood by one of skill in the art, the chemical synthesis of amonosaccharide may be achieved using well-established procedures incarbohydrate chemistry; however, monosaccharides for use as startingcompounds in the disclosed synthesis schemes may be obtained from avariety of commercial vendors and chemically modified by one of skill inthe art to arrive at the immunogenic synthetic construct of the instantinvention, e.g., according to the synthesis schemes disclosed herein.Published chemical modifications include, but are not limited to, themethod for the synthesis of 4-methoxyphenyl-α-D-galactopyranosideproposed in Comfort, et al., Biochem. 46: 3319-3330 (2007.) Briefly,4-methoxyphenyl-α-D-galactopyranoside may be synthesized fromD-galactose by acetylation, glycosidation with 4-methoxyphenol, followedby Zemplén deacetylation according to published methods. (Montgomery etal. (1942) J. Am. Chem. Soc. 64, 690-694.)

Similarly, various strategies for the synthesis and introduction ofMeOPN to a monosaccharide are familiar to one of skill in the art. Aparticular method is described in C. Mara et al, Bioorg. Med. Chem.Lett. 6180-6183 (2011.) This reference describes the reaction with ethyldichlorophosphate followed by reaction with protected amines.

As discussed above, the synthetic construct of the instant invention maybe chemically activated in order to add one or more chemical linkinggroup(s) capable of reacting with a carrier protein. As contemplatedherein, the activation of a construct of the instant invention may beperformed according to conventional methods familiar to one of skill inthe art. Such methods include, e.g., the use of cyanylating reagentssuch as I-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP);carbodiimides, hydrazides, active esters, p-nitrobenzoic acid,N-hydroxysuccinimide, and trimethylsilyl trifluoromethanesulfonate(TMSOTf.) Activating the construct may also be achieved by reacting thesaccharide with 2, 2, 6,6-tetramethylpiperidin-1-oxyl (TEMPO.) See,e.g., US Pub. No. 2014/0141032.

While the immunogenic synthetic constructs of the instant invention maybe administered to a subject in an unconjugated form, it is contemplatedherein that upon synthesis, the construct may be chemically activatedand chemically conjugated in vitro to one or more carrier molecules,e.g., one or more T cell-dependent carrier proteins prior toadministration in order to provide an enhanced immune response. Indeed,as appreciated by one of skill in the art, children are only capable ofmounting an IgM response in the face of polysaccharide antigens; adultsare capable of generating an IgG, IgA and IgM response. Thus, by linkinga carrier protein to the synthetic construct, the immune responsetriggered in vivo by the construct will change from a T-cell independentresponse to one which is T-cell dependent. As such, the immune responsethat is triggered is enhanced and thus markedly different than whatmight otherwise be produced in vivo by an unconjugated construct.

In a particular embodiment, the carrier molecule is a carrier protein.As used herein, a “carrier protein” refers to a protein, or an analog orfragment thereof, which ideally contains at least one T-cell epitope.Suitable carrier proteins for use with the instant invention arefamiliar to one of skill in the art and are commercially availableand/or may be created and purified by one of skill in the art usingconventional methods. For example, carrier proteins for use with theinstant invention include bacterial toxins that are immunologicallyeffective carriers and that have been rendered safe by chemical orgenetic means for administration to a subject. Examples include, but arenot limited to, inactivated bacterial toxins such as diphtheria toxoid,CRM₁₉₇, tetanus toxoid, pertussis toxoid, E. coli heat labileenterotoxin (LT), the binding component of E. coli heat labileenterotoxin (LTB), E. coli adhesins and/or fimbriae, and exotoxin A fromPseudomonas aeruginosa. Bacterial outer membrane proteins such as, e.g.,outer membrane complex c (OmpC), porins, transferrin binding proteins,pneumococcal surface protein A (PspA), pneumococcal adhesin protein(PsaA), or pneumococcal surface proteins BVH-3 and BVH-11 can also beused. Other proteins, such as protective antigen (PA) of Bacillusanthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serumalbumin, bovine serum albumin (BSA) and purified protein derivative oftuberculin (PPD) can also be used.

In a particular embodiment, the carrier protein is selected from thegroup consisting of inactivated bacterial toxins, bacterial outermembrane proteins, protective antigen (PA) of Bacillus anthracis,ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovineserum albumin (BSA) and purified protein derivative of tuberculin (PPD.)In a particular embodiment, the inactivated bacterial toxin is selectedfrom the group consisting of diphtheria toxoid, cross-reactive material197 (CRM₁₉₇), tetanus toxoid, pertussis toxoid, the binding component ofE. coli heat labile enterotoxin (LTB), E. coli adhesins and/or fimbriae,and exotoxin A from Pseudomonas aeruginosa. In a particular embodiment,the carrier protein is the inactivated bacterial toxin CRM₁₉₇. Inanother particular embodiment, the bacterial outer membrane protein isselected from the group consisting of outer membrane complex c (OmpC),porins, transferrin binding proteins, pneumococcal surface protein A(PspA), pneumococcal adhesin protein (PsaA), pneumococcal surfaceprotein BVH-3, and pneumococcal surface protein BVH-11. Such carrierproteins are available from a variety of commercial vendors.

It is also contemplated herein that proteins from ETEC may be used ascarrier molecules. Possible ETEC protein carriers include, but are notlimited to, the B subunit of the heat labile enterotoxin, and fimbrialsubunits. The latter includes subunits of various ETEC colonizationfactors such as, e.g., Cfal (CfaE and/or CfaB), CS6 (CssB and/or CssA),CS3 (CstG and/or CstH), CS17 (CsbA and/or CsbD) and CSI (CooA.) Furtherexamples of ETEC proteins and details regarding the use of ETEC proteinsas possible carrier molecules can be found, e.g., in US 2015/0258201 A1,the entire contents of which are incorporated by reference herein.

As contemplated herein, a carrier protein may be linked to more than onesynthetic construct in order to enhance the immunogenicity of theconstruct against C. jejuni. In one embodiment, multiple syntheticMeOPN-6-Gal constructs are linked to a single carrier protein. In aparticular embodiment, a conjugate vaccine of the instant inventioncomprising a MeOPN-6-Gal: CRM₁₉₇ ratio (w/w) of at least 8:1 or more isenvisioned herein.

After conjugation, free and conjugated saccharide constructs can beseparated using a variety of conventional methods. Purification methodsare familiar to one of skill in the art and include, e.g.,ultrafiltration, size exclusion chromatography, density gradientcentrifugation, hydrophobic interaction chromatography, and/or ammoniumsulfate fractionation.

Possible methods of conjugating an activated monosaccharide orsaccharide construct of the instant invention to a carrier protein arefamiliar to one of skill in the art and include, e.g., reductiveamination of a monosaccharide involving the coupling of the resultingamino group with one end of an adipic acid linker group, and thencoupling a protein to the other end of the adipic acid linker group;cyanylation conjugation, wherein the saccharide construct is activatedeither by cyanogens bromide (CNBr) or by1-cyano-4-dimethylammoniumpyridinium tetrafluoroborate (CDAP) tointroduce a cyanate group to the hydroxyl group, which forms a covalentbond to the amino or hydrazide group upon addition of the proteincomponent; and a carbodiimide reaction, wherein carbodiimide activatesthe carboxyl group on one component of the conjugation reaction, and theactivated carbonyl group reacts with the amino or hydrazide group on theother component. If necessary, these reactions may also be employed toactivate the components of the carrier protein prior to the conjugationreaction. As contemplated herein, in a particular embodiment, a processinvolving the introduction of amino groups into the monosaccharide(e.g., by replacing terminal ═O groups with —NH₂) followed byderivatization with an adipic diester (e.g., adipic acidN-hydroxysuccinimido diester) and reaction with carrier protein may beused.

It is also contemplated herein that the synthetic construct may belinked directly to the carrier protein. Direct linkages to the proteinmay comprise oxidation of the monosaccharide followed by reductiveamination with the protein using conventional methods.

The synthetic constructs of the instant invention, e.g., comprising oneor more MeOPN-6-Gal monosaccharides, may further comprise one or moreadditional saccharides, as well as one or more additional chemicalcompounds or moieties or fragments or derivatives thereof. A variety ofchemical compounds can serve as a chemical backbone to link the variouscomponents of an immunogenic synthetic construct of the instantinvention, and/or to link the synthetic construct as a whole to one ormore carrier proteins. Compounds that may be used to make a polymericconstruct or conjugate include, e.g., modified starch moieties,cyclodextrin, and nigeran.

As particularly contemplated herein, the construct may compriseadditional saccharides, moieties, or compounds which may be incorporatedfor a variety of reasons, e.g., to increase the chemical stability ofthe synthetic construct and/or to enhance the delivery orbioavailability of the construct. In a particular embodiment, it iscontemplated herein that additional saccharides, moieties, and compoundsmay be chemically associated with one or more MeOPN-6-Gal constructseither directly or indirectly through one or more linkers or othercompounds, in order to enhance the immunogenicity of the syntheticconstruct against C. jejuni in a subject. Thus, additional saccharidesfor use in a synthetic construct of the instant invention include, butare not limited to, monosaccharides present in the capsule of various C.jejuni strains, e.g., galactose or other modified forms thereof,including MeOPN-2-Gal, fructose, glucose, heptose N-acetylgalactosamine, N-acetyl glucosamine, glucitol, glucose or modified formsor derivatives thereof, including monosaccharides containing one or moreMeOPN moieties, including but not limited to MeOPN-2-Gal andMeOPN-6-Gal. Such saccharides may be used in an amount and incombination with one or more MeOPN-6-Gal monosaccharides which mayenhance the immunogenicity of the synthetic construct against C. jejuni.For example, FIG. 1 lists the CPS repeating blocks and specific heptoseunits and MeOPN linkages of C. jejuni serotype complexes HS1, HS3, HS4,and HS23/36.

In view of the foregoing, as provided in the below examples, FIG. 15depicts a synthetic polymeric construct which comprises more than oneMeOPN-6-Gal monosaccharide; FIG. 18 depicts a synthetic polymericconstruct which comprises more than one MeOPN-6-Gal monosaccharide andalso comprises additional monosaccharides MeOPN-2-Gal and MeOPN-1-Fru.It is contemplated herein that the presence of these additionalcomponents in a construct or conjugate of the instant invention willenhance the immunogenicity of the construct or conjugate against C.jejuni.

As understood herein, “associated” includes any manner of chemicalcombination, e.g., the synthetic construct may comprise severalsynthetic MeOPN-6-Gal monosaccharides chemically joined in a chain as apolymer, or in various combinations with any number of one or more othersaccharides. Such construct may be further conjugated to a carrierprotein.

As contemplated herein, the methods of the instant invention aredirected to inducing an immune response against C. jejuni in a subjectcomprising administering an effective amount of the immunogenicsynthetic construct to the subject. In particular embodiments, theconstruct is administered to the subject in the form of a compositioncomprising the synthetic construct as an active pharmaceuticalingredient, e.g., a pharmaceutical composition, more particularly, as avaccine formulation comprising the synthetic construct linked to acarrier protein. Thus, as used herein, an “effective amount” can referto the amount of the immunogenic synthetic construct alone or in acomposition, including in a pharmaceutical composition comprising one ormore other active pharmaceutical agents or excipients.

Moreover, as understood herein, an “effective amount” refers to animmunologically effective amount of the immunogenic synthetic construct(conjugated or unconjugated) suitable to elicit an immune response inthe subject. As discussed above, an “immune response” encompassestriggering a humoral and/or cellular immune response in the subject. Asa result, a meaningful clinical benefit to the subject is provided. Suchbenefit may be, e.g., preventing, ameliorating, treating, inhibiting,and/or reducing one of more pathological conditions associated withcampylobacteriosis or related sequelae. Thus, the methods of the presentinvention can be considered therapeutic methods or preventative orprophylactic methods. In a particular embodiment, it is contemplatedherein that the immunogenic synthetic construct and/or conjugate of theinstant invention may be administered to a subject and thus preventdiarrhea caused by C. jejuni in the subject.

One of skill in the art will appreciate that the administration of thesynthetic construct of the instant invention encompasses the use of theconstructs and/or the compositions, e.g., vaccine formulations, of theinstant invention to generate immunity in a subject if later challengedby infection with C. jejuni. It is further understood herein, however,that the synthetic constructs, conjugates, compositions, vaccineformulations and methods of the present invention do not necessarilyprovide total immunity to C. jejuni and/or totally cure or eliminate alldisease symptoms.

Suitable effective amounts of the immunogenic synthetic constructs ofthe instant invention can be readily determined by those of skill in theart and will depend upon the age, weight, species (if non-human) andmedical condition of the subject to be treated, and whether theconstruct is administered in a conjugated or unconjugated form. Forexample, initial information may be gleaned in laboratory experiments,and an effective amount for humans subsequently determined throughconventional dosing trials and routine experimentation. As contemplatedherein, an effective amount of the construct or conjugate forvaccination against C. jejuni infection may be from between about 1 μgor less to about 100 μg or more per kg body weight. As a general guide,a suitable amount of a construct or conjugate of the invention can be anamount between from about 0.1 μg to about 10 mg per dosage amount withor without an adjuvant. Moreover, immunization comprising administeringone or more boosting doses may be performed using between from about 0.1μg to about 10 mg per dose with or without adjuvant.

It is contemplated herein that the constructs and compositions of theinstant invention may be administered to a subject by a variety ofroutes according to conventional methods, including but not limited toparenteral (e.g., by intracistemal injection and infusion techniques),intradermal, transmembranal, transdermal (including topical),intramuscular, intraperitoneal, intravenous, intra-arterial,intralesional, subcutaneous, oral, and intranasal (e.g., inhalation)routes of administration. Administration can also be by continuousinfusion or bolus injection.

In addition, the compositions of the instant invention can beadministered in a variety of dosage forms. These include, e.g., liquidpreparations and suspensions, including, preparations for parenteral,subcutaneous, intradermal, intramuscular, intraperitoneal or intravenousadministration (e.g., injectable administration), such as sterileisotonic aqueous solutions, suspensions, emulsions or viscouscompositions that may be buffered to a selected pH. In a particularembodiment, it is contemplated herein that the constructs andcompositions of the instant invention are administered to a subject asan injectable, including but not limited to injectable compositions fordelivery by intramuscular, intravenous, subcutaneous, or transdermalinjection. Such compositions may be formulated using a variety ofpharmaceutical excipients, carriers or diluents familiar to one of skillin the art.

In another particular embodiment, the synthetic immunogenic constructand compositions of the instant invention may be administered orally.Oral formulations for administration according to the methods of thepresent invention may include a variety of dosage forms, e.g.,solutions, powders, suspensions, tablets, pills, capsules, caplets,sustained release formulations, or preparations which are time-releasedor which have a liquid filling, e.g., gelatin covered liquid, wherebythe gelatin is dissolved in the stomach for delivery to the gut. Suchformulations may include a variety of pharmaceutically acceptableexcipients described herein, including but not limited to mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose, andmagnesium carbonate.

In a particular embodiment, it is contemplated herein that a compositionfor oral administration may be a liquid formulation. Such formulationsmay comprise a pharmaceutically acceptable thickening agent which cancreate a composition with enhanced viscosity which facilitates mucosaldelivery of the immunogen, e.g., by providing extended contact with thelining of the stomach. Such viscous compositions may be made by one ofskill in the art employing conventional methods and employingpharmaceutical excipients and reagents, e.g., methylcellulose, xanthangum, carboxymethyl cellulose, hydroxypropyl cellulose, and carbomer.

Other dosage forms suitable for nasal or respiratory (mucosal)administration, e.g., in the form of a squeeze spray dispenser, pumpdispenser or aerosol dispenser, are contemplated herein. Dosage formssuitable for rectal or vaginal delivery are also contemplated herein.The constructs, conjugates, and compositions of the instant inventionmay also be lyophilized and may be delivered to a subject with orwithout rehydration using conventional methods.

As understood herein, the methods of the instant invention compriseadministering the immunogenic synthetic construct to a subject accordingto various regimens, i.e., in an amount and in a manner and for a timesufficient to provide a clinically meaningful benefit to the subject.Suitable administration regimens for use with the instant invention maybe determined by one of skill in the art according to conventionalmethods. For example, it is contemplated herein that an effective amountmay be administered to a subject as a single dose, a series of multipledoses administered over a period of days, or a single dose followed by aboosting dose thereafter, e.g., several years later. The term “dose” or“dosage” as used herein refers to physically discrete units suitable foradministration to a subject, each dosage containing a predeterminedquantity of the synthetic construct and/or conjugate as the activepharmaceutical ingredient calculated to produce a desired response.

The administrative regimen, e.g., the quantity to be administered, thenumber of treatments, and effective amount per unit dose, etc. willdepend on the judgment of the practitioner and are peculiar to eachsubject. Factors to be considered in this regard include physical andclinical state of the subject, route of administration, intended goal oftreatment, as well as the potency, stability, and toxicity of theparticular construct, conjugate or composition. As understood by one ofskill in the art, a “boosting dose” may comprise the same dosage amountas the initial dosage, or a different dosage amount. Indeed, when aseries of immunizations is administered in order to produce a desiredimmune response in the subject, one of skill in the art will appreciatethat in that case, an “effective amount” may encompass more than oneadministered dosage amount.

As contemplated herein, the compositions of the instant invention, andparticularly pharmaceutical compositions and vaccines of the instantinvention, are preferably sterile and contain an amount of the constructand/or conjugate vaccine in a unit of weight or volume suitable foradministration to a subject. The volume of the composition administeredto a subject (dosage unit) will depend on the method of administrationand is discernible by one of skill in the art. For example, in the caseof an injectable, the volume administered typically may be between 0.1and 1.0 ml, preferably approximately 0.5 ml.

As understood herein, a “pharmaceutical composition” of the instantinvention comprises an immunogenic synthetic construct (unconjugated orconjugated to a carrier protein or combination thereof) in combinationwith one or more pharmaceutically acceptable excipients, carriers, ordiluents. The term “pharmaceutically acceptable” is used to refer to anon-toxic material that is compatible with a biological system such as acell, cell culture, tissue, or organism.

Examples of pharmaceutically acceptable excipients, carriers anddiluents are familiar to one of skill in the art and can be found, e.g.,in Remington's Pharmaceutical Sciences (latest edition), Mack PublishingCompany, Easton, Pa. For example, pharmaceutically acceptable excipientsinclude, but are not limited to, wetting or emulsifying agents, pHbuffering substances, binders, stabilizers, preservatives, bulkingagents, adsorbents, disinfectants, detergents, sugar alcohols, gellingor viscosity enhancing additives, flavoring agents, and colors.Pharmaceutically acceptable carriers include macromolecules such asproteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, trehalose, lipidaggregates (such as oil droplets or liposomes), and inactive virusparticles. Pharmaceutically acceptable diluents include, but are notlimited to, water, saline, and glycerol.

As understood by one of skill in the art, the type and amount ofpharmaceutically acceptable additional components included in thepharmaceutical compositions of the instant invention may vary, e.g.,depending upon the desired route of administration and desired physicalstate, solubility, stability, and rate of in vivo release of thecomposition. For example, for administration by intravenous, cutaneous,subcutaneous, or other injection, a vaccine formulation is typically inthe form of a pyrogen-free, parenterally acceptable aqueous solution ofsuitable pH and stability, and may contain an isotonic vehicle as wellas pharmaceutical acceptable stabilizers, preservatives, buffers,antioxidants, or other additives familiar to one of skill in the art.

In a particular embodiment, pharmaceutical compositions in the form of avaccine formulation comprising the immunogenic synthetic constructand/or conjugate of the instant invention, alone or in combination withother active agents and/or pharmaceutically acceptable excipients, arecontemplated for administration to a subject as provided herein. Bothmonovalent vaccines (e.g., designed to immunize against a single antigenor single microorganism), and polyvalent vaccines (e.g., designed toimmunize against two or more strains of the same microorganism, oragainst two or more microorganisms) are contemplated herein. In oneembodiment, a vaccine formulation of the instant invention is apolyvalent formulation. In a particular embodiment, the vaccineformulations of the instant invention may be a polyvalent formulationagainst one or more strains of C. jejuni, including but not limited to,serotypes HS 23/36, HS1, HS2, HS3, HS4, and HS5/31. It is alsocontemplated herein that a polyvalent formulation of the instantinvention may be directed against one or more strains of C. jejuniand/or other bacterial strain including those which haveMeOPN-containing capsules.

The formulation of the vaccines of the present invention can beaccomplished using art recognized methods. For example, in addition toan immunologically effective amount of the construct or conjugatevaccine, a “vaccine formulation” of the instant invention may furthercomprise one or more non-immunogenic components, e.g., one or morepharmaceutically acceptable excipients, carriers, diluents, stabilizers,preservatives, buffers, and disinfectants as discussed above. To thisend, one of skill in the art will appreciate that the development of arobust and stable vaccine formulation will ideally employ variousexcipients and formulation parameters that will provide stability to theantigen and thus prevent aggregation, loss of protein structure, and/orchemical degradation such as oxidation and deamidation. One of skill inthe art using routine experimentation and conventional methods candetermine the particular pH, buffers, and stabilizers that are wellsuited for the development of robust and stable vaccine formulations ofthe instant invention. See, e.g., Morefield, G. (2011) The APPS Journal,13: 191-200.

In addition, the pharmaceutical compositions, and particularly thevaccine formulations of the instant invention, may further comprise oneor more adjuvants. As understood by one of skill in the art, an adjuvantis a substance that aids a subject's immune response to an antigen. Anadjuvant can be used to increase the immunogenic efficacy of a vaccine,and may also have the ability to increase the stability of a vaccineformulation. Thus, faster and longer lasting immune responses may bepossible in vivo through the addition of an adjuvant to a vaccineformulation. Adjuvants suitable for use with the compositions of theinstant invention are familiar to one of skill in the art and areavailable from a variety of commercial vendors. These include, forexample, glycolipids; chemokines; compounds that induce the productionof cytokines and chemokines; interferons; inert carriers, such as alum,bentonite, latex, and acrylic particles; pluronic block polymers; depotformers; surface active materials, such as saponin, lysolecithin,retinal, liposomes, and pluronic polymer formulations; macrophagestimulators, such as bacterial lipopolysaccharide; alternate pathwaycomplement activators, such as insulin, zymosan, endotoxin, andlevamisole; non-ionic surfactants; poly(oxyethylene)-poly(oxypropylene)tri-block copolymers; trehalose dimycolate (TDM); cell wall skeleton(CWS); complete Freund's adjuvant; incomplete Freund's adjuvant;macrophage colony stimulating factor (M-CSF); tumor necrosis factor(TNF); 3-O-deacylated MPL; CpG oligonucleotides; polyoxyethylene ethers,polyoxyethylene esters, aluminum,Poly[di(carboxylatophenoxy)phosphazene](PCPP), monophosphoryl lipid A,QS-21, cholera toxin and formyl methionyl peptide.

In one embodiment, the adjuvant may be selected from the groupconsisting of antigen delivery systems (e.g. aluminum compounds orliposomes), immunopotentiators (e.g. toll-like receptor ligands), or acombination thereof (e.g., AS01 or ASO4.) These substances are familiarto one of skill in the art. In a particular embodiment, an adjuvant foruse in the compositions and methods of the instant invention is selectedfrom the group consisting of toll-like receptor ligands, aluminumphosphate, aluminum hydroxide, monophosphoryl lipid A, liposomes, andderivatives and combinations thereof. See, e.g., Alving, C. et al.,2012, Expert Rev Vaccines 11, 733-44; Alving, C. et al. (2012) Curr OpinImmunol 24, 310-5; Alving C. and Rao, M, (2008) Vaccine 26, 3036-3045;U.S. Pat. No. 6,090,406; U.S. Pat. No. 5,916,588.

In addition to the immunogenic synthetic construct and/or conjugate, thecompositions of the instant invention may further comprise one or moreother active pharmaceutical ingredient, including but not limited to,additional immunoregulatory agents. As understood herein, animmunoregulatory agent is a substance that can induce, potentiate,activate or otherwise stimulate the immune system of the subject. Theseimmunoregulatory agents include, for example, substances selected fromthe group consisting of antigens of one or more strains of C. jejuni,antigens of ETEC, Shigella lipopolysaccharide structures, andunconjugated carrier proteins. (See, e.g., US 2015/0258201 A1.)

In addition, the compositions and vaccines of the instant invention maybe administered alone or in combination with other vaccines, and/orother therapeutic or immunoregulatory agents. Such additional vaccinesand agents may be administered to a subject in any manner, e.g., before,after, or concurrently with the immunogenic synthetic constructs andcompositions of the instant invention.

The invention also provides a kit comprising the immunogenic syntheticconstructs and/or compositions of the instant invention. In a particularembodiment, the kit may comprise the conjugate vaccine and instructionsfor administering the conjugate vaccine to a subject. The kit canoptionally also contain one or more other therapeutic orimmunoregulatory agents. The kit can optionally contain one or morediagnostic tools and instructions for use. For example, a compositioncomprising two or more vaccines can be included, or separatepharmaceutical compositions containing different vaccines or therapeuticagents. The kit can also contain separate doses of the conjugate vaccinefor serial or sequential administration. The kit can contain suitabledelivery devices, e.g., syringes, inhalation devices, and the like,along with instructions for administrating the compositions. The kit canoptionally contain instructions for storage, reconstitution (ifapplicable), and administration of any or all therapeutic agentsincluded. The kits can include a plurality of containers reflecting thenumber of administrations to be given to a subject. If the kit containsa first and second container, then a plurality of these can be present.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodiments,and examples provided herein, are merely illustrative of the principlesand applications of the present invention. It is therefore to beunderstood that numerous modifications can be made to the illustrativeembodiments and examples, and that other arrangements can be devisedwithout departing from the spirit and scope of the present invention asdefined by the appended claims. All patent applications, patents,literature and references cited herein are hereby incorporated byreference in their entirety.

EXAMPLES Example 1 Synthesis of p-Methoxyphenyl and AminopentylGlycosides of the MeOPN→6-Gal Construct: MeOPN→6-α-D-Galp-(1→OMP andMeOPN→6-β-D-Galp-(1→O(CH₂)₅NH₂

Previously, using conventional methods and mass spectrometry, wedetected a non-stoichiometric MeOPN unit at the 2 position of galactose(MeOPN-2-Gal) in C. jejuni 81-176 CPS, with a ³¹P resonance similar tothat depicted in FIG. 21A (peak Y) (Kanipes M I, et al. (2006.) J.Bacteriol. 188:3273-3279.). We confirmed this MeOPN-2-Gal linkage by NMR(FIG. 22A) through the detection of a cross-peak between the ³¹Presonance Y (δ_(P) 14.45) of MeOPN and H-2 (δ_(H) 4.52) of the galactoseunit in a ¹H-³¹P correlation experiment. In some 81-176 CPSpreparations, albeit of lower intensity, the ³¹P NMR spectrum displayedan additional resonance at δ_(P) 14.15 (designated peak Z) (FIG. 21B.) Asimilar peak was also observed in another 81-176 CPS preparation (amutant in gene CJJ81176_1420) that exhibited a cross-peak between thephosphorous of MeOPN and H-6 resonances of some of the CPS galactoseunits, which resonated very near the methyl resonances of MeOPN (δ_(H)3.75 to 3.81) (FIG. 22B.) The NMR data suggested that peak Z in 81-176corresponded to a non-stoichiometric placement of MeOPN at position 6 ofgalactose (MeOPN-6-Gal.). These data and additional genetic studies aredescribed in greater detail in Example 8 below.

In order to test the potential of a prototype synthetic monosaccharideanti-C. jejuni vaccine, p-methoxyphenyl and aminopentyl glycosides ofMeOPN→6-Gal constructs, i.e., MeOPN→6-α-D-Galp-(1→OMP andMeOPN→6-J-D-Galp-(1→O(CH₂)₅NH₂, respectively, were synthesized.Specifically, as provided below and as depicted in FIG. 2 and FIG. 3,MeOPN→6-α-D-Galp construct may be synthesized as the p-methoxyphenyl(OMP) glycoside, MeOPN→6-α-D-Galp-(1→OMP (FIG. 2, Scheme 1) and thenequipped with an aminopentyl linker at C-1 (as the β anomer)MeOPN→6-β-D-Galp-(1→O(CH₂)₅NH₂ for conjugation to a carrier protein(FIG. 3, Scheme 2.)

Summary Synthesis of MeOPN→6-α-D-Galp-(1→OMP (FIG. 2, Scheme 1)

Since MeOPN can be readily removed in mild acidic media, a suitablesynthetic strategy circumventing such conditions was needed. As astarting compound, 4-methoxyphenyl-α-D-galactopyranoside was synthesizedaccording to published methods. (See, Comfort, et al., Biochem.46:3319-3330 (2007.)) Briefly, 4-methoxyphenyl-α-D-galactopyranoside wassynthesized from D-galactose by acetylation, glycosidation with4-methoxyphenol, followed by Zemplén deacetylation according topublished methods. (Montgomery et al. (1942) J. Am. Chem. Soc. 64,690-694).

Starting from 4-methoxyphenyl-α-D-galactopyranoside (compound 1), atrityl group was selectively introduced to the 6-position. Originally,benzoylation was performed on compound 2, but the extensive migrationobserved during the introduction of MeOPN required the elucidation of amore suitable protecting group. Allyl groups were thus selected toprotect the C-2, C-3 and C-4 positions which were resistant tomigration. The allyl groups were later deprotected with catalytichydrogenolysis, yielding compound 3, which proved to be compatible withthe MeOPN modification. Next, the trityl group was removed givingcompound 4 exposing 6-OH for modification.

The strategy for the introduction of MeOPN is similar to a publishedreaction. (See Mara et al. Bioorg. Med. Chem. Lett. 6180-6183 (2011.)Compound 4 was treated with commercially available methyldichlorophosphate in the presence of triethyl amine, followed byammonolysis. Due to the dual chiral nature of the newly introducedMeOPN, product 5 was collected as a mixture of two diastereoisomers. ³¹PNMR was able to confirm that product 5 was indeed a 1:1 mixture of twodiastereoisomers, revealing two phosphorus signals at 10.5 ppm. ¹H NMRalso revealed two sets of signals with two anomeric and two OCH₃ signals(data not shown.)

The reaction yielded a mixture of side products, the most abundant beingthe replacement of the O-methyl group by a second NH₂. Removal of theallyl group with palladium (II) chloride generated product 6. Similar tocompound 5, a mixture of diastereoisomers was observed by ¹H and ³¹P NMR(FIG. 13). A 2D ¹H-³¹P HMBC NMR experiment was able to confirm that theMeOPN was introduced to the O-6 position, showing correlation signalsbetween phosphorous with both H-6 signals and OCH₃.

Summary Synthesis of MeOPN→6-β-D-Galp-(1→O(CH₂)₅NH₂ (FIG. 3, Scheme 2)

After successfully designing a strategy for the MeOPN modification, theconstruct was joined to a linker in order to make a vaccine conjugate.First, the 4-methoxyphenyl (OMP) was removed from galactoside (compound3 in FIG. 2.) The corresponding hemiacetal was converted into thetrichloroacetimidate donor (compound 7). The5-amino-N-phthalimido-pentanyl linker was then introduced with TMSOTf asthe activator at 0° C. Compound 8 was collected with 65% in the β and29% in the a anomer. The removal of trityl group afforded compound 9with a free hydroxyl group for the introduction of MeOPN. Using theprocedure described above, phosphoramidate (compound 10) was collectedas a mixture of two diastereoisomers. Allyl and phthalimido protectinggroups were subsequently removed giving compound 11 and then compound12.

Materials and Methods:

The compounds were synthesized using conventional methods and allchemicals were purchased from commercial suppliers and used as received.Molecular sieves were activated by heating with a heating mantle underreduced pressure. Thin layer chromatography (TLC) was carried out on TLCsilica gel F₂₅₄. Sugar compounds were visualized by UV light or bycharring with 10% H₂SO₄ in ethanol. Flash chromatography was performedwith silica gel P60, (43-60 μm, 230-400 mesh.) ¹H NMR and ¹³C NMRspectra were recorded with Bruker 400 or 600 MHz spectrometers (BrukerDaltonics Inc, Billerica, Mass.) The proton signal of residual,non-deuterated solvent (δ 7.24 ppm for CHCl₃) was used as internalreference for ¹H spectra. For ¹³C spectra, the chemical shifts arereported relative to the solvent (δ 77.1 ppm for CDCl₃.) Chemical shiftsare reported in parts per million (ppm.) Coupling constants are reportedin Hertz (Hz.) The following abbreviations are used to indicate themultiplicities: s, singlet; d, doublet; t, triplet; m, multiplet.Optical rotations were measured on a Rudolph Research Autopol IIIautomatic polarimeter (Rudolph Research Analytical, Hackettstown, N.J.)and concentration (c) is expressed in g/100 ml. High-resolution massspectra for the synthetic compounds were recorded by electron sprayionization mass spectroscopy (time of flight analyzer.)

4-Methoxyphenyl 6-O-trityl-α-D-galactopyranoside (compound 2)

To a solution of compound 1 (2.7 g, 9.3 mmol) dissolved in pyridine (40mL), trityl chloride (3.1 g, 11 mmol) was added and the reaction mixturewas stirred at 60° C. for 3 days. The reaction mixture was thenconcentrated and purified with flash chromatography (1:1 EtOAc-hexanes)to yield compound 2 (4.7 g, 95%.) ¹H NMR (400 MHz, CDCl₃): δ 7.44-7.20(m, 15H, Ar—H); 7.11-6.83 (m, 4H, MeOC₆H₄); 5.51 (d, 1H, J=3.6 Hz, H-1);4.05-3.93 (m, 4H, H-2, H-3, H-4, H-5); 3.79 (s, 3H, OCH₃); 3.54-3.32 (m,2H, H-6.) ¹³C NMR (100 MHz, CDCl₃): δ 155.3, 151.2, 150.6, 144.3, 143.8,143.7, 143.6, 129.1, 128.6, 128.0, 127.9, 127.8, 127.5, 127.3, 127.1,127.0, 118.5, 117.9, 114.6, 114.5, 114.4 (Ar); 98.4 (C-1); 87.0, 71.2,70.0, 69.3 (C-2, C-3, C-4, C-5); 63.6 (C-6); 55.6 (CH₃.) HRMS (ESI):Calcd. For C₃₂H₃₂O₇ [M+Na]⁺: 551.2046. found: 551.2021.

4-Methoxyphenyl 2,3,4-tri-O-allyl-6-O-trityl-α-D-galactopyranoside(compound 3)

A solution of compound 2 (4.7 g, 8.8 mmol) dissolved in DMF (60 mL) withallyl bromide (4.6 mL, 53 mmol) was cooled to 0° C. Sodium hydride, 60%dispersion in mineral oil (1.2 g, 29 mmol) was added and the reactionmixture was stirred for 1 h at 0° C. The reaction was then quenched withMeOH (10 mL), poured into ice-cold water (100 mL) and extracted withEtOAc (3×100 mL.) The organic layer was dried over Na₂SO₄ andconcentrated. Purification by flash chromatography eluting with 1:7EtOAc-hexanes gave compound 3 (5.1 g, 89%.) [α]_(D) ²⁵=+132.6° (c+0.1,CHCl₃); ¹H NMR (400 MHz, CDCl₃): δ7.38-7.18 (m, 15H, Ar—H); 7.10-6.75(m, 4H, MeOC₆H₄); 6.00-5.53 (m, 3H, CH₂—CH═CH₂); 5.42 (d, 1H, J=3.2 Hz,H-1); 5.33-4.98 (m, 6H, CH₂—CH═CH₂); 4.37-3.72 (m, 13H, CH₂—CH═CH₂, H-2,H-3, H-4, H-5, OCH₃); 3.38 (m, 1H, H-6a); 3.01 (m, 1H, H-6b.) ¹³C NMR(100 MHz, CDCl₃): δ 155.0, 151.0, 143.9 (Ar); 135.2, 135.1, 135.0(CH₂—CH═CH₂); 128.6, 127.8, 127.0, 119.0, 117.4, 117.3, 116.4, 114.4(CH₂—CH═CH₂, Ar); 97.5 (C-1); 86.8; 78.2 (C-2); 77.4 (C-4); 76.1 (C-5);73.9, 72.5, 71.9 (CH₂—CH═CH₂); 70.4 (C-3) 63.3 (C-6); 55.6 (OCH₃.) HRMS(ESI): Calcd. For C₄₁H₄₄O₇ [M+Na]⁺: 671.2985. found: 671.2970.

4-Methoxyphenyl 2,3,4-tri-O-allyl-α-D-galactopyranoside (compound 4)

A solution of compound 3 (300 mg, 0.46 mmol) in 80% aqueous AcOH (5 mL)was stirred at 80° C. for 1.5 h. The reaction mixture was concentratedbefore purification by flash chromatography (1:6 EtOAc-hexanes) givingcompound 4 (147 mg, 78%.) ¹H NMR (400 MHz, CDCl₃): δ 7.02-6.78 (m, 4H,MeOC₆H₄); 5.95-5.89 (m, 3H, CH₂—CH═CH₂); 5.50 (d, 1H, J=3.5 Hz, H-1);5.35-5.12 (m, 6H, CH₂—CH═CH₂); 4.42 (dd, 1H, J₁=3.2 Hz, J₂=9.3 Hz, H-3);4.27-3.89 (m, 10H, CH₂—CH═CH₂, H-2, H-4, H-5, OH); 3.81 (m, 1H, H-6a);3.74 (s, 3H, OCH₃); 3.70 (m, 1H, H-6b.) ¹³C NMR (100 MHz, CDCl₃): δ155.1, 150.9 (Ar); 135.0, 134.9 (CH₂—CH═CH₂); 118.6, 118.0, 117.4,116.6, 114.5 (CH₂—CH═CH₂, Ar); 97.5 (C-1); 78.2, 75.9, 74.0, 72.6, 72.0,71.0 (CH₂—CH═CH₂, C-2, C-3, C-4, C-5); 62.7 (C-6); 55.6 (OCH₃.) HRMS(ESI): Calcd. For C₂₂H₃₀O₇ [M+Na]⁺: 429.1890. found: 429.1891.

4-Methoxyphenyl2,3,4-tri-O-allyl-6-O-methyl-phosphramidate-α-D-galactopyranoside(compound 5)

To a solution of compound 4 (65 mg, 0.16 mmol) and methyldichlorophosphate (150 μL, 1.3 mmol) dissolved in CH₂Cl₂ (3 mL) withmolecular sieves, Et₃N (175 μL, 1.3 mmol) was added drop-wise. Thereaction mixture was stirred at room temperature for 5 hours. Uponcompletion of the reaction as judged by TLC, ammonia gas was injectedinto the reaction mixture through a needle. After 10 min, the reactionmixture was filtered and concentrated. Purification with columnchromatography (1:1 EtOAc-hexanes) yielded compound 5 (15 mg, 19%.) ¹HNMR (400 MHz, CDCl₃): δ 7.04-6.77 (m, 4H, MeOC₆H₄); 5.99-5.85 (m, 3H,CH₂—CH═CH₂); 5.48 (2d, 1H, J=3.6 Hz, H-1); 5.36-5.10 (m, 6H,CH₂—CH═CH₂); 4.41 (m, 1H, H-3); 4.29-4.10 (m, 8H, CH₂—CH═CH₂, H-2, H-4);3.95-3.86 (m, 3H, H-5, H-6); 3.73 (s, 3H, OCH₃); 3.57 (2d, 3H, J=11.4Hz, OCH₃); 2.75, 2.56 (2d, 2H, NH₂.) ¹³C NMR (100 MHz, CDCl₃): δ 155.2,155.0, 150.9 (Ar); 135.0, 134.9 (CH₂—CH═CH₂); 128.9, 128.3, 118.8,118.5, 117.7, 117.5, 117.4, 116.6, 114.5, 114.4 (CH₂—CH═CH₂, Ar); 97.6,97.2 (C-1); 78.1, 75.8, 74.4, 74.0, 72.7, 71.9, 70.5, 70.4, 70.0, 69.9,68.5, 65.5, (CH₂—CH═CH₂, C-2, C-3, C-4, C-5, C-6); 55.7, 53.3, 53.2(OCH₃.) HRMS (ESI): Calcd. For C₂₃H₃NO₉P [M+H]⁺: 500.2050. found:500.2035.

4-Methoxyphenyl 6-O-methyl-phosphoramidate-α-D-galactopyranoside(compound 6)

To a solution of compound 5 (17.0 mg) dissolved in MeOH (1 mL), PdCl₂(5.0 mg) was added and the reaction mixture was stirred at roomtemperature for 3 h. The reaction mixture was then filtered andconcentrated. Purification with column chromatography (pure EtOAc)yielded compound 6 (5.1 mg, 39%.) ¹H NMR (400 MHz, D₂O): δ6.98-6.80 (m,4H, MeOC₆H₄); 5.39 (2d, 1H, J=3.6 Hz, H-1); 4.13 (m, 1H, H-3); 4.01-3.85(m, 4H, H-4, H-5, H-6); 3.78 (m, 1H, H-2); 3.63 (OCH₃); 3.41 (2d, 3H,J=11.4 Hz, OCH₃.) ¹³C NMR (100 MHz, D₂O): δ154.6, 150.0, 149.9, 119.3,119.1, 114.9 (Ar); 98.1, 97.9 (C-1); 70.3, 70.2, 70.0, 69.1, 68.8, 67.8,65.8 (C-2, C-3, C-4, C-5, C-6); 55.6 (OCH₃); 53.6, 53.5, 53.4 (OCH₃.)HRMS (ESI): Calcd. For C14H23NO9P [M+H]⁺ cal. 380.1111. found 380.1110.

2,3,4-Tri-O-allyl-6-O-trityl-α,β-D-galactopyranosyl trichloroacetimidate(compound 7)

To a solution of compound 3 (5.0 g, 7.7 mmol) dissolved in CH₃CN (480mL) and H₂O (120 mL), cerium ammonium nitrate (12.8 g, 23 mmol) wasadded and the reaction mixture was stirred for 20 min at 0° C. Themixture was then diluted with brine (200 mL) and extracted with EtOAc(3×300 mL.) The organic layer was washed with saturated aq. Na₂CO₃ andwater, dried over Na₂SO₄, concentrated and purified with columnchromatography (1:6 EtOAc-hexanes.) The resulting hemiacetal (3.3 g, 6.1mmol) was dissolved in anhydrous CH₂Cl₂ (120 ml) and CCl₃CN (310 μL, 30mmol) and K₂CO₃ (420 mg, 30 mmol) were added. The reaction mixture wasstirred at room temperature overnight before it was filtered throughCelite® and concentrated. Purification with flash chromatography (1:4EtOAc-hexanes with 1% Et₃N by volume) gave compound 7 as an α,β-mixture(3.6 g, 57% over 2 steps) (compounds 7A and 7B.)

7A: ¹H NMR (400 MHz, CDCl₃): δ 8.52 (s, 1H, NH); 7.42-7.18 (m, 15H,Ar—H); 6.46 (d, 1H, J=3.6 Hz, H-1); 6.00-5.61 (m, 3H, CH₂—CH═CH₂);5.39-4.98 (m, 6H, CH₂—CH═CH₂); 4.32-3.84 (m, 10H, CH₂—CH═CH₂, H-2, H-3,H-4, H-5); 3.35 (m, 1H, H-6a); 3.09 (m, 1H, H-6b); ¹³C NMR (100 MHz,CDCl₃): δ 161.3, 160.8, 143.9, 143.7, 135.2, 135.0, 134.9, 134.8, 134.1,133.8, 128.8, 128.6, 127.8, 127.1, 127.0 (Ar, CH₂—CH═CH₂); 117.9, 117.4,117.3, 116.7, 116.5 (CH₂—CH═CH₂); 104.0 (C-1); 86.8 (C-3); 86.7 (C-2);83.8 (C-3); 82.6; 76.7 (C-4); 75.3, 74.1, 72.5, 72.2, 71.8, 71.0(CH₂—CH═CH₂, C-5); 61.9 (C-6.) HRMS (ESI): Calcd. For C₃₆H₃₈Cl₃NO₆[M+Na]⁺: 708.1663. found: 708.1673.

7B: ¹H NMR (400 MHz, CDCl₃): δ 8.59 (s, 1H, NH); 7.41-7.18 (m, 15H,Ar—H); 5.90 (m, 2H, CH₂—CH═CH₂); 5.62 (m, 2H, H-1, CH₂—CH═CH₂);5.35-5.01 (m, 6H, CH₂—CH═CH₂); 4.31-3.83 (m, 6H CH₂—CH═CH₂); 3.83 (m,1H, H-5); 3.76 (dd, 1H, J=8.2 Hz, J₂=9.7 Hz, H-3); 3.62 (t, 1H, J=5.9Hz, H-2), 3.48-3.39 (m, 2H, H-4, H-6a); 3.12 (dd, 1H, J₁=7.2 Hz, J₂=9.3Hz, H-6b.) ¹³C NMR (100 MHz, CDCl₃): δ 161.5, 143.8 (Ar); 135.4, 134.9,134.8 (CH₂—CH═CH₂); 128.7, 128.6, 128.0, 127.9, 127.1 (Ar); 117.3,117.0, 116.8 (CH₂—CH═CH₂); 98.5 (C-1); 86.8 (C-2); 81.6 (C-3); 77.8(C-5); 74.6 (C-3) 74.2, 73.8, 73.3, 72.0 (CH₂—CH═CH₂, C-4); 62.4 (C-6.)HRMS (ESI): Calcd. For C₃₆H₃₈Cl₃NO₆ [M+Na]⁺: 708.1663. found: 708.1673.

5-Amino-N-phthalimido-pentanyl2,3,4-tri-O-allyl-6-O-trityl-β-D-galactopyranoside(compound 8)

Trichloroacetimidate (compound 7, both anomers) (1.1 g, 1.6 mmol) and5-amino-N-phthalimido-pentanol (560 mg, 2.4 mmol) were dissolved inanhydrous CH₂Cl₂ (25 mL) and the reaction mixture was cooled to 0° C.TMSOTf (15 μL, 0.080 mmol) was added drop-wise and the reaction mixturewas stirred for 15 min at 0° C. The reaction was then neutralized withEt₃N (15 μL) and concentrated. Purification with flash chromatography(1:8 EtOAc-hexanes) gave compound 8 (783 mg, 65%.) ¹H NMR (400 MHz,CDCl₃): δ 7.80-7.67 (m, 4H, phthalimido protons); 7.41-7.19 (m, 15H,Ar—H); 5.98-5.59 (m, 3H, CH₂—CH═CH₂); 5.33-4.94 (m, 6H, CH₂—CH═CH₂);4.30-3.84 (m, 8H, CH₂—CH═CH₂, H-1, linker-CHH)); 3.77 (d, 1H, J=2.9 Hz,H-5); 3.62 (t, 2H, J=7.3 Hz, linker-CH₂); 3.45-3.35 (m, 4H, H-2, H-4,H-6a, linker —CHH); 3.29 (dd, 1H, J₁=3.0 Hz, J₂=9.8 Hz, H-3); 3.13 (dd,1H, J=9.4 Hz, J₂=10.1 Hz, H-6b); 1.65 (m, 4H, linker-CH₂); 1.40 (m, 2H,linker-CH₂.) ¹³C NMR (100 MHz, CDCl₃): 168.4, 143.8 (Ar); 135.7, 135.3,135.2 (CH₂—CH═CH₂); 133.9, 132.1, 128.7, 127.9, 127.1, 123.2 (Ar);116.8, 116.5 (CH₂—CH═CH₂); 103.7 (C-1); 86.8; 81.5 (C-1); 79.2 (C-2);73.9, 73.6, 73.4, 73.3 (C-5, C-4, CH₂—CH═CH₂); 71.9, 69.4 (linker); 62.5(C-6); 37.9, 29.2, 28.4, 23.4 (linker.) HRMS (ESI): Calcd. For C₄₇H₅₁NO₈[M+Na]⁺: 780.3513. found 780.3489.

5-Amino-N-phthalimido-pentanyl 2,3,4-tri-O-allyl-β-D-galactopyranoside(compound 9)

A solution of compound 8 (493 mg, 0.65 mmol) dissolved in 80% aqueousAcOH (10 mL) was stirred at 80° C. for 1 h. The reaction mixture wasconcentrated before purification by flash chromatography (1:1EtOAc-hexanes) giving compound 9 (260 mg, 78%.) ¹H NMR (400 MHz, CDCl₃):δ 7.81-7.66 (m, 4H, phthalimido protons); 5.92-5.82 (m, 3H, CH₂—CH═CH₂);5.30-5.10 (m, 6H, CH₂—CH═CH₂); 4.37-4.02 (m, 6H, CH₂—CH═CH₂); 4.22 (d,1H, J=7.7 Hz, H-1); 3.88 (m, 2H, H-6a, linker-CHH); 3.69-3.60 (m, 4H,H-4, H-6b, linker-CH₂); 3.51-3.42 (m, 2H, H-2, linker-CHH); 3.39 (m, 1H,H-5); 3.28 (dd, 1H, J₁=3.0 Hz, J₂=9.8 Hz, H-3); 2.09 (m, 1H, 6-OH); 1.65(m, 4H, linker-CH₂); 1.40 (m, 2H, linker-CH₂.) ¹³C NMR (100 MHz, CDCl₃):δ 168.5 (phthalimido C═O); 135.3, 135.0, 133.9 (CH₂—CH═CH₂); 132.1,123.2 (phthalimido); 117.8, 116.7, 116.6 (CH₂—CH═CH₂); 103.9 (C-1); 81.6(C-3); 79.1 (C-2); 74.6 (C-5) 74.0 (C-4); 73.7, 73.6 (CH₂—CH═CH₂); 72.1,69.6 (linker); 62.5 (C-6); 37.8, 29.2, 28.3, 23.3 (linker.) HRMS (ESI):Calcd. For C₂₈H₃₇NO₈ [M+Na]⁺: 538.2417. found 538.2403.

5-Amino-N-phthalimido-pentanyl2,3,4-tri-O-allyl-6-O-methylphosphramidate-β-D-galactopyranoside(compound 10)

To a solution of compound 9 (400 mg, 0.78 mmol) and methyldichlorophosphate (0.70 mL, 6.0 mmol) dissolved in CH₂Cl₂ (15 mL) withmolecular sieves, Et₃N (0.70 mL, 5.0 mmol) was added drop-wise. Thereaction mixture was stirred at room temperature for 12 hours. Uponcompletion of the reaction as judged by TLC, ammonia gas was injectedinto the reaction mixture through a needle. After 10 min, the reactionmixture was filtered and concentrated. Purification with columnchromatography (9:1 EtOAc-MeOH) yielded compound 10 (129 mg, 27%.) ¹HNMR (400 MHz, CDCl₃): 7.80-7.68 (phthalimido protons); 5.88 (m, 3H,CH₂—CH═CH₂); 5.30-5.10 (m, 6H, CH₂—CH═CH₂); 4.23-4.10 (m, 9H,CH₂—CH═CH₂, H-1, linker-CH₂); 3.82 (m, 1H, H-5); 3.71-3.39 (m, 9H, OCH₃,H-4, H-2, H-6a, H-6b, linker-CH₂); 3.28 (m, 1H, H-3); 2.87 (dd, 2H,J₁=5.3 Hz J₂=13.0 Hz, NH₂); 1.66 (m, 4H, linker-CH₂); 1.38 (m, 2H,linker-CH₂.) ¹³C NMR (100 MHz, CDCl₃): δ 168.5 (Ar); 135.4, 135.2, 134.9(CH₂—CH═CH₂); 133.9, 132.1, 123.2 (Ar); 117.5, 117.2, 116.8, 116.7,116.6 (CH₂—CH═CH₂); 103.8 (C-1); 81.4 (C-3); 78.9 (C-2); 74.0, 73.8,73.3, 73.2, 73.0, 72.9, 72.1 (CH₂—CH═CH₂, C-5, C-4); 69.8, 69.7 (C-6)65.3; 65.0, 64.9 (linker) 53.4, 53.3 (OCH₃); 37.9, 29.7, 29.2, 28.3(linker.) HRMS (ESI): Calcd. For C₂₉H₄₁N₂O₁₀P [M+H]⁺: 609.2578. found609.2585.

5-Amino-N-phthalimido-pentanyl6-O-methylphosphramidate-β-D-galactopyranoside(compound 11)

To a solution of compound 10 (95 mg, 0.16 μmol) dissolved in MeOH (4mL), PdCl₂ (20 mg) was added and the reaction mixture was stirred atroom temperature for 4 h. The reaction mixture was then filtered andconcentrated. Purification with column chromatography (9:1 EtOAc-MeOH)gave compound 11 (57 mg, 75%.) ¹H NMR (400 MHz, D₂O): δ7.64 (m, 4H,phthalimido protons); 4.23 (d, 1H, J=8.0 Hz, H-1); 4.01 (m, 2H, H-6);3.78-3.70 (m, 3H, H-4, H-5, linker-CHH)); 3.59-3.45 (m, 7H, OCH₃,linker-CH₂ linker-CHH, H-3); 3.33 (dd, 1H, J₁=8.0 Hz, J₂=9.8 Hz, H-2);1.51 (m, 4H, linker-CH₂); 1.22 (m, 2H, linker-CH₂.) ¹³C NMR (100 MHz,D₂O): 170.9, 134.5, 133.9, 131.3, 126.0, 123.1 (Ar); 102.6 (C-1); 73.2(C-5); 72.5 (C-3); 71.9 (C-2); 70.3, 70.2 (linker); 68.1 (C-4); 65.4(C-6); 53.6 (OCH₃); 48.7; 37.6 (linker); 28.2; 27.2, 22.3 (linker.) HRMS(ESI): Calcd. For C₂₀H₂₉N₂O₁₀P [M+H]⁺: 489.1639. found 489.1624.

5-Amino-pentanyl 6-O-methylphosphramidate-β-D-galactopyranoside(compound 12)

To a solution of compound 11 (23 mg, 0.047 μmol) dissolved in 95% EtOH(1 mL), hydrazine monohydrate (16 μL, 0.33 μmol) was added and thereaction mixture was stirred at room temperature overnight. The reactionmixture was then concentrated and purification with columnchromatography (3:1 EtOAc-MeOH) gave compound 12 (14 mg, 82%.) ¹H NMR(400 MHz, D₂O): δ4.27 (d, 1H, J=7.1 Hz, H-1); 4.03 (m, 2H, linker-CH₂);3.81-3.75 (m, 3H, H-4, H-5, H-6a); 3.61-3.48 (m, 5H, OCH₃, H-3, H-6b);3.36 (dd, 1H, J₁=7.9, J₂=9.9 Hz, H-2); 2.82 (t, 2H, J=7.5 Hz,linker-CH₂); 1.52 (m, 4H, linker-CH₂); 1.30 (m, 2H, linker-CH₂.) ¹³C NMR(100 MHz, D₂O): δ 102.6 (C-1); 73.2 (C-5); 72.5 (C-3); 70.5 (C-2); 70.1(C-6); 68.1 (C-4); 60.0 (linker); 48.7 (OCH₃); 39.2, 28.0, 26.3, 22.0,21.9 (linker.) HRMS (ESI): Calcd. For C₁₂H₂₇N₂O₈P [M+H]⁴: 359.1584.found 359.1587.

The synthesis of the structure MeOPN→6-β-D-Galp-(1→O(CH₂)₅NH₂ can alsobe depicted as set forth in FIG. 4, Scheme 2a, and is summarized below:

Starting from a previously reported compound (Comfort, et al., Biochem.46: 3319-3330 (2007)), 4-methoxyphenyl-α-D-galactopyranoside (see Scheme2a, compound 1), trityl group was selectively introduced to C-6.Originally, benzoylation was performed on compound 2 (Scheme 2a,compound 2), however, extensive migration observed during theintroduction of MeOPN lead to the elucidation of a more suitableprotecting group. Therefore, allyl groups were selected to protect theC-2, C-3 and C-4 positions which were resistant to migration. Allylgroups were later deprotected with catalytic hydrogenolysis which provedto be compatible with the MeOPN modification.

After allyl groups were installed, an amino-pentanyl linker wasintroduced to the anomeric position as a site for conjugation. Startingfrom galactoside (Scheme 2a, compound 3), 4-methoxyphenyl group (OMP)was first removed with cerium ammonium nitrate (CAN.) The correspondinghemiacetal was then converted into trichloroacetimidate donor (seeScheme 2a, compound 4.) 5-Amino-N-phthalimido-pentanyl linker was thenintroduced with TMSOTf as activator at 0° C. Compound 5 was collectedwith 65% as the β anomer and 29% as the a anomer. The removal of tritylgroup gave compound 6 with a free 6-hydroxyl group for modification.

The strategy for the introduction of MeOPN group is similar to areaction proposed by C. Mara et al, Bioorg. Med. Chem. Lett. 6180-6183(2011.) Compound 6 was treated with commercially available methyldichlorophosphate in the presence of triethyl amine, followed byammonolysis. Due to the chirality nature of the newly introduced MeOPN(R and S), compound 7 was collected as a mixture of twodiastereoisomers. ¹H NMR was able to confirm that compound 7 was indeeda 1:1 mixture of two diastereoisomers, revealing two sets of signalsthroughout the spectrum, such can be seen for anomeric and O-Me signals.The reaction yielded a mixture of side products, the most abundant beingthe O-Me group being replaced by a second NH₂, accounting for the pooryield of this reaction.

Allyl and phthalimido protecting groups were removed with palladium (II)chloride and hydrazine respectively, generating compound 8 and compound9. Similar to compound 7, a mixture of diastereoisomers is apparent inNMR. Although not optically pure, the ³¹P NMR result agrees with nativeMeOPN-containing polysaccharides, having a phosphorous signals around 14ppm. ³¹H-³¹P HMBC NMR experiment was able to confirm that the MeOPN wasintroduced to the O-6 position, showing correlation signal with O-Me aswell as the H-6 signals (data not shown.)

The details of the above synthesis of MeOPN→6-β-D-Galp-(1→O(CH₂)₅NH₂ isprovided below and in Scheme 2a:

4-Methoxyphenyl 6-O-trityl-α-D-galactopyranoside (Scheme 2a, compound 2)

To a solution of compound 1 (2.7 g, 9.3 mmol) dissolved in pyridine (40mL), trityl chloride (3.1 g, 11 mmol) was added and the reaction mixturewas stirred at 60° C. for 3 days. The reaction mixture was thenconcentrated and purified with flash chromatography (1:1 EtOAc-hexanes)to yield compound 2 (4.7 g, 95%.) [α]_(D) ²⁵=+91.2° (c=0.21, CHCl₃); ¹HNMR (400 MHz, CDCl₃): δ 7.44-7.20 (m, 15H, Ar—H); 7.11-6.83 (m, 4H,MeOC₆H₄); 5.51 (d, 1H, J=3.6 Hz, H-1); 4.05-3.93 (m, 4H, H-2, H-3, H-4,H-5); 3.79 (s, 3H, OCH₃); 3.54-3.32 (m, 2H, H-6.) ¹³C NMR (100 MHz,CDCl₃): δ 155.3, 151.2, 150.6, 144.3, 143.8, 143.7, 143.6, 129.1, 128.6,128.0, 127.9, 127.8, 127.5, 127.3, 127.1, 127.0, 118.5, 117.9, 114.6,114.5, 114.4 (Ar); 98.4 (C-1); 87.0, 71.2, 70.0, 69.3 (C-2, C-3, C-4,C-5); 63.6 (C-6); 55.6 (CH₃.) HRMS (ESI): Calcd. For C₃₂H₃₂O₇ [M+Na]⁺:551.2046. found: 551.2021.

4-Methoxyphenyl 2,3,4-tri-O-allyl-6-O-trityl-α-D-galactopyranoside(Scheme 2a, compound 3)

A solution of compound 2 (4.7 g, 8.8 mmol) dissolved in DMF (60 mL) withallyl bromide (4.6 mL, 53 mmol) was cooled to 0° C. Sodium hydride, 60%dispersion in mineral oil (1.2 g, 29 mmol) was added and the reactionmixture was stirred for 1 h at 0° C. The reaction was then quenched withMeOH (10 mL), poured into ice-cold water (100 mL) and extracted withEtOAc (3×100 mL.) The organic layer was dried over Na₂SO₄ andconcentrated. Purification by flash chromatography eluting with 1:7EtOAc-hexanes gave compound 3 (see scheme 1, structure 3) (5.1 g, 89%.)¹H NMR (400 MHz, CDCl₃): δ 7.38-7.18 (m, 15H, Ar—H); 7.10-6.75 (m, 4H,MeOC₆H₄); 6.00-5.53 (m, 3H, CH₂—CH═CH₂); 5.42 (d, 1H, J=3.2 Hz, H-1);5.33-4.98 (m, 6H, CH₂—CH═CH₂); 4.37-3.72 (m, 13H, CH₂—CH═CH₂, H-2, H-3,H-4, H-5, OCH₃); 3.38 (m, 1H, H-6a); 3.01 (m, 1H, H-6b.) ¹³C NMR (100MHz, CDCl₃): δ 155.0, 151.0, 143.9 (Ar); 135.2, 135.1, 135.0(CH₂—CH═CH₂); 128.6, 127.8, 127.0, 119.0, 117.4, 117.3, 116.4, 114.4(CH₂—CH═CH₂, Ar); 97.5 (C-1); 86.8; 78.2 (C-2); 77.4 (C-4); 76.1 (C-5);73.9, 72.5, 71.9 (CH₂—CH═CH₂); 70.4 (C-3) 63.3 (C-6); 55.6 (OCH₃.) HRMS(ESI): Calcd. For C₄₁H₄₄O₇ [M+Na]⁺: 671.2985. found: 671.2970.

2,3,4-Tri-O-allyl-6-O-trityl-α,β-D-galactopyranosyl trichloroacetimidate(Scheme 2a, compound 4)

To a solution of compound 3 (5.0 g, 7.7 mmol) dissolved in CH₃CN (480mL) and H₂O (120 mL), cerium ammonium nitrate (12.8 g, 23 mmol) wasadded and the reaction mixture was stirred for 20 min at 0° C. Themixture was then diluted with brine (200 mL) and extracted with EtOAc(3×300 mL.) The organic layer was washed with saturated aq. Na₂CO₃ andwater, dried over Na₂SO₄, concentrated and purified with columnchromatography (1:6 EtOAc-hexanes.) The resulting hemiacetal (3.3 g, 6.1mmol) was dissolved in anhydrous CH₂Cl₂ (120 ml) and CCl₃CN (310 μL, 30mmol) and K₂CO₃ (420 mg, 30 mmol) were added. The reaction mixture wasstirred at room temperature overnight before it was filtered throughCelite® and concentrated. Purification with flash chromatography (1:4EtOAc-hexanes with 1% Et₃N by volume) gave compound 4 as an α,β-mixture(3.6 g, 57% over 2 steps.)

5-Amino-N-phthalimido-pentanyl2,3,4-tri-O-allyl-6-O-trityl-β-D-galactopyranoside (Scheme 2a, compound5)

Trichloroacetimidate (compound 4) (1.1 g, 1.6 mmol) and5-amino-N-phthalimido-pentanol (560 mg, 2.4 mmol) were dissolved inanhydrous CH₂Cl₂ (25 mL) and the reaction mixture was cooled to 0° C.TMSOTf (15 μL, 0.080 mmol) was added drop-wise and the reaction mixturewas stirred for 15 min at 0° C. The reaction was then neutralized withEt₃N (15 μL) and concentrated. Purification with flash chromatography(1:8 EtOAc-hexanes) gave compound 5 (783 mg, 65%.) ¹H NMR (400 MHz,CDCl₃): δ 7.80-7.67 (m, 4H, phthalimido protons); 7.41-7.19 (m, 15H,Ar—H); 5.98-5.59 (m, 3H, CH₂—CH═CH₂); 5.33-4.94 (m, 6H, CH₂—CH═CH₂);4.30-3.84 (m, 8H, CH₂—CH═CH₂, H-1, linker-CHH); 3.77 (d, 1H, J=2.9 Hz,H-5); 3.62 (t, 2H, J=7.3 Hz, linker-CH₂); 3.45-3.35 (m, 4H, H-2, H-4,H-6a, linker —CHH); 3.29 (dd, 1H, J₁=3.0 Hz, J₂=9.8 Hz, H-3); 3.13 (dd,1H, J=9.4 Hz, J₂=10.1 Hz, H-6b); 1.65 (m, 4H, linker-CH₂); 1.40 (m, 2H,linker-CH₂.) ¹³C NMR (100 MHz, CDCl₃): 168.4, 143.8 (Ar); 135.7, 135.3,135.2 (CH₂—CH═CH₂); 133.9, 132.1, 128.7, 127.9, 127.1, 123.2 (Ar);116.8, 116.5 (CH₂—CH═CH₂); 103.7 (C-1); 86.8; 81.5 (C-1); 79.2 (C-2);73.9, 73.6, 73.4, 73.3 (C-5, C-4, CH—CH═CH₂); 71.9, 69.4 (linker); 62.5(C-6); 37.9, 29.2, 28.4, 23.4 (linker.) HRMS (ESI): Calcd. For C₄₇H₅₁NO₈[M+Na]⁺: 780.3513. found 780.3489.

5-Amino-N-phthalimido-pentanyl 2,3,4-tri-O-allyl-β-D-galactopyranoside(Scheme 2a, compound 6)

A solution of compound 5 (493 mg, 0.65 mmol) dissolved in 80% aqueousAcOH (10 mL) was stirred at 80° C. for 1 h. The reaction mixture wasconcentrated before purification by flash chromatography (1:1EtOAc-hexanes) giving compound 6 (260 mg, 78%.) ¹H NMR (400 MHz, CDCl₃):δ 7.81-7.66 (m, 4H, phthalimido protons); 5.92-5.82 (m, 3H, CH₂—CH═CH₂);5.30-5.10 (m, 6H, CH₂—CH═CH₂); 4.37-4.02 (m, 6H, CH₂—CH═CH₂); 4.22 (d,1H, J=7.7 Hz, H-1); 3.88 (m, 2H, H-6a, linker-CHH); 3.69-3.60 (m, 4H,H-4, H-6b, linker-CH₂); 3.51-3.42 (m, 2H, H-2, linker-CHH); 3.39 (m, 1H,H-5); 3.28 (dd, 1H, J=3.0 Hz, J₂=9.8 Hz, H-3); 2.09 (m, 1H, 6-OH); 1.65(m, 4H, linker-CH₂); 1.40 (m, 2H, linker-CH₂.) ¹³C NMR (100 MHz, CDCl₃):δ 168.5 (phthalimido C═O); 135.3, 135.0, 133.9 (CH₂—CH═CH₂); 132.1,123.2 (phthalimido); 117.8, 116.7, 116.6 (CH₂—CH═CH₂); 103.9 (C-1); 81.6(C-3); 79.1 (C-2); 74.6 (C-5) 74.0 (C-4); 73.7, 73.6 (CH₂—CH═CH₂); 72.1,69.6 (linker); 62.5 (C-6); 37.8, 29.2, 28.3, 23.3 (linker.) HRMS (ESI):Calcd. For C₂₈H₃₇NO [M+Na]⁺: 538.2417. found 538.2403.

5-Amino-N-phthalimido-pentanyl2,3,4-tri-O-allyl-6-O-methylphosphoramidate-β-D-galactopyranoside(Scheme 2a, compound 7)

To a solution of compound 6 (400 mg, 0.78 mmol) and methyldichlorophosphate (0.70 mL, 6.0 mmol) dissolved in CH₂Cl₂ (15 mL) withmolecular seives, Et₃N (0.70 mL, 5.0 mmol) was added drop-wise. Thereaction mixture was stirred at room temperature for 12 hours. Uponcompletion of the reaction as judged by TLC, ammonia gas was injectedinto the reaction mixture through a needle. After 10 min, the reactionmixture was filtered and concentrated. Purification with columnchromatography (9:1 EtOAc-MeOH) yielded product 7 (129 mg, 27%.) ¹H NMR(400 MHz, CDCl₃): 7.80-7.68 (phthalimido protons); 5.88 (m, 3H,CH₂—CH—CH₂); 5.30-5.10 (m, 6H, CH₂—CH═CH₂); 4.23-4.10 (m, 9H,CH₂—CH═CH₂, H-1, linker-CH₂); 3.82 (m, 1H, H-5); 3.71-3.39 (m, 9H, OCH₃,H-4, H-2, H-6a, H-6b, linker-CH₂); 3.28 (m, 1H, H-3); 2.87 (dd, 2H,J₁=5.3 Hz J₂=13.0 Hz, NH₂); 1.66 (m, 4H, linker-CH₂); 1.38 (m, 2H,linker-CH₂.) ¹³C NMR (100 MHz, CDCl₃): δ 168.5 (Ar); 135.4, 135.2, 134.9(CH₂—CH═CH₂); 133.9, 132.1, 123.2 (Ar); 117.5, 117.2, 116.8, 116.7,116.6 (CH₂—CH═CH₂); 103.8 (C-1); 81.4 (C-3); 78.9 (C-2); 74.0, 73.8,73.3, 73.2, 73.0, 72.9, 72.1 (CH₂—CH═CH₂, C-5, C-4); 69.8, 69.7 (C-6)65.3; 65.0, 64.9 (linker) 53.4, 53.3 (OCH₃); 37.9, 29.7, 29.2, 28.3(linker.) HRMS (ESI): Calcd. For C₂₉H₄₁N₂O₁₀P [M+H]⁺: 609.2578. found609.2585.

5-Amino-N-phthalimido-pentanyl6-O-methylphosphoramidate-1-D-galactopyranoside(Scheme 2a, compound 8)

To a solution of compound 7 (95 mg, 0.16 μmol) dissolved in MeOH (4 mL),PdCl₂ (20 mg) was added and the reaction mixture was stirred at roomtemperature for 4 h. The reaction mixture was then filtered andconcentrated. Purification with column chromatography (9:1 EtOAc-MeOH)gave compound 8 (57 mg, 75%.) ¹H NMR (400 MHz, D₂O): δ7.64 (m, 4H,phthalimido protons); 4.23 (d, 1H, J=8.0 Hz, H-1); 4.01 (m, 2H, H-6);3.78-3.70 (m, 3H, H-4, H-5, linker-CHH)); 3.59-3.45 (m, 7H, OCH₃,linker-CH₂ linker-CHH, H-3); 3.33 (dd, 1H, J=8.0 Hz, J₂=9.8 Hz, H-2);1.51 (m, 4H, linker-CH₂); 1.22 (m, 2H, linker-CH₂.) ¹³C NMR (100 MHz,D₂O): 170.9, 134.5, 133.9, 131.3, 126.0, 123.1 (Ar); 102.6 (C-1); 73.2(C-5); 72.5 (C-3); 71.9 (C-2); 70.3, 70.2 (linker); 68.1 (C-4); 65.4(C-6); 53.6 (OCH₃); 48.7; 37.6 (linker); 28.2; 27.2, 22.3 (linker.) HRMS(ESI): Calcd. For C₂₀H₂₉N₂O₁₀P [M+H]⁺: 489.1639. found 489.1624.

5-Amino-pentanyl 6-O-methylphosphoramidate-β-D-galactopyranoside (Scheme2a, compound 9)

To a solution of compound 8 (23 mg, 0.047 μmol) dissolved in 95% EtOH (1mL), hydrazine monohydrate (16 μL, 0.33 μmol) was added and the reactionmixture was stirred at room temperature overnight. The reaction mixturewas then concentrated and purification with column chromatography (3:1EtOAc-MeOH) gave compound 9 (14 mg, 82%.) ¹H NMR (400 MHz, D₂O): δ4.27(d, 1H, J=7.1 Hz, H-1); 4.03 (m, 2H, linker-CH₂); 3.81-3.75 (m, 3H, H-4,H-5, H-6a); 3.61-3.48 (m, 5H, OCH₃, H-3, H-6b); 3.36 (dd, 1H, J=7.9,J₂=9.9 Hz, H-2); 2.82 (t, 2H, J=7.5 Hz, linker-CH₂); 1.52 (m, 4H,linker-CH₂); 1.30 (m, 2H, linker-CH₂.) ¹³C NMR (100 MHz, D₂O): δ102.6(C-1); 73.2 (C-5); 72.5 (C-3); 70.5 (C-2); 70.1 (C-6); 68.1 (C-4); 60.0(linker); 48.7 (OCH₃); 39.2, 28.0, 26.3, 22.0, 21.9 (linker.) HRMS(ESI): Calcd. For C₁₂H₂₇N₂O₈P [M+H]⁺: 359.1584. found 359.1587.

Example 2 Synthesis of MeOPN→+2-β-D-Galp-(1→OMP Summary Synthesis ofMeOPN→2-β-D-Galp-(1→OMP (FIG. 5, Scheme 3)

The synthesis of MeOPN→2-β-D-Galp-(1→OMP is depicted in FIG. 5, scheme3. The synthesis of galactoside (product 7) began with a known compound,4-methoxyphenyl 3,4-O-isopropylidene-6-O-trityl-β-D-galactopyranoside(product 1), which was prepared from D-galactose following publishedprocedures (Scheme 1.) (Comfort D A, et al., Biochem 2007;46:3319-3330.) To distinguish the C-2 position, O-allylation wasperformed generating product 2 in excellent yield. Since MeOPN can beremoved by acidic media, suitable protecting groups needed to beinstalled. Thus, O-isopropylidene and O-trityl groups were removedgiving product 3, which was then per-benzoylated affording product 4.Next, the allyl group was removed yielding a free 2-OH for modification.The introduction of MeOPN group to product 5 followed a strategypreviously developed in our lab, involving first a phosphorylation withcommercially available methyl dichlorophosphate followed by ammonolysis.(Jiao, Y. et al., Carbohydr. Res. (2015) doi:10.1016/j.carres.2015.09.012). The ³¹P NMR spectrum of product 5revealed two phosphorus signals of roughly 1:1 ratio due to theformation of two diastereoisomers. Product 6 was de-benzoylatedfurnishing O-Me-phosphoramidate galactoside product 7. Interestingly, wewere able to purify one of the diastereoisomers using flashchromatography. ³¹P NMR spectrum of the diastereoisomer 7* revealed asingle signal at 14.27 ppm (see FIG. 14.)

Materials and Methods:

Conventional methods were used to synthesize the compounds, and allchemicals were purchased from commercial suppliers and used as received.Molecular sieves were activated by heating with a heating mantle underreduced pressure. Thin layer chromatography (TLC) was carried out on TLCsilica gel F₂₅₄. Sugar compounds were visualized by UV light or bycharring with 10% H₂SO₄ in ethanol. Flash chromatography was performedwith silica gel P60, (43-60 μm, 230-400 mesh.) ¹H NMR and ¹³C NMRspectra were recorded with Bruker 400 or 600 MHz spectrometers (BrukerDaltonics Inc, Billerica, Mass.) The proton signal of residual,non-deuterated solvent (δ 7.24 ppm for CHCl₃) was used as internalreference for ¹H spectra. For ¹³C spectra, the chemical shifts arereported relative to the solvent (δ 77.1 ppm for CDCl₃.) Chemical shiftsare reported in parts per million (ppm.) Coupling constants are reportedin Hertz (Hz.) The following abbreviations are used to indicate themultiplicities: s, singlet; d, doublet; t, triplet; m, multiplet.Optical rotations were measured on a Rudolph Research Autopol IIIautomatic polarimeter and concentration (c) is expressed in g/100 ml.High-resolution mass spectra for the synthetic compounds were recordedby electron spray ionization mass spectroscopy (time of flightanalyzer.)

4-Methoxyphenyl2-O-allyl-3,4-O-isopropylidene-6-O-trityl-β-D-galactopyranoside (product2)

A solution of product 1 (0.68 g, 1.2 mmol) dissolved in DMF (18 mL) withallyl bromide (0.16 mL, 1.8 mmol) was cooled to 0° C. Sodium hydride,60% dispersion in mineral oil (57 mg, 1.4 mmol) was added and thereaction mixture was stirred for 1 h at 0° C. The reaction was thenquenched with MeOH (2 mL), poured into ice-cold water (40 mL) andextracted with CH₂Cl₂ (3×50 mL.) The organic layer was dried over Na₂SO₄and concentrated. Purification by flash chromatography eluting with 1:7EtOAc-hexanes gave 2 (0.69 g, 95%.) [α]_(D) ²⁵=+40.2° (c=0.05, CHCl₃);¹H NMR (400 MHz, CDCl₃): δ 7.46-7.19 (m, 15H, Ar); 7.10-6.75 (m, 4H,MeOC₆H₄); 5.92 (m, 1H, CH₂—CH═CH₂); 5.34-5.19 (m, 2H, CH₂—CH═CH₂); 4.67(d, 1H, J=8.1 Hz, H-1); 4.36 (m, 2H, CH₂—CH═CH₂); 4.08 (m, 2H, H-3,H-4); 3.73 (s, 3H, OCH₃); 3.61-3.53 (m, 3H, H-2, H-5, H-6a); 3.34 (m,1H, H-6b); 1.47 (s, 3H, CH₃); 1.29 (s, 3H, CH₃.) ¹³C NMR (100 MHz,CDCl₃): δ 155.2, 151.5, 144.0, 143.9 (Ar); 134.9 (CH₂—CH═CH₂); 128.8,127.9, 127.8, 127.0, 126.9, 118.6, 118.3, 117.7, 117.4, 114.5, 114.4,110.2, 109.3 (CH₂—CH═CH₂, Ar); 102.2 (C-1); 86.8 (CMe₂) 79.4 (C-2);79.2; (C-3); 73.8 (C-4); 72.9 (CH₂—CH═CH₂); 72.6 (C-5); 63.0 (C-6); 55.6(OCH₃); 27.9, 26.3 (CH₃.) HRMS (ESI): Calcd. For C₃₈H₄₀NaO₇ [M+Na]⁺:631.2672. found: 631.2670.

4-Methoxyphenyl 2-O-allyl-β-D-galactopyranoside (product 3)

A solution of product 2 (0.69 g, 1.1 mmol) in 80% aqueous AcOH (10 mL)was stirred at 80° C. for 1 h. The reaction mixture was concentratedunder reduced pressure. Purification by flash chromatography (1:1EtOAc-hexanes) gave 3 (0.35 g, 94%.) [α]_(D) ²⁵=+90.2° (c=0.2, CHCl₃);¹H NMR (400 MHz, CDCl₃): δ 7.01-7.78 (m, 4H, MeOC₆H₄); 5.91 (m, 1H,CH₂—CH═CH₂); 5.19 (m, 2H, CH₂—CH═CH₂); 4.83 (d, 1H, J=7.5 Hz, H-1);4.53-4.25 (m, 2H, CH₂—CH═CH₂); 4.14 (m, 1H, H-5); 3.96 (m, 1H, H-6a);3.85 (m, 1H, H-6b); 3.76 (s, 3H, OCH₃); 3.62 (m, 3H, H-2, H-3, H-4.) ¹³CNMR (100 MHz, CDCl₃): δ 155.4, 151.1 (Ar); 134.5 (CH₂—CH═CH₂); 118.5,118.2, 118.0, 114.6, 114.6 (CH₂—CH═CH₂, Ar); 102.6 (C-1); 78.4 (C-3);75.9 (C-4); 73.7 (CH₂—CH═CH₂); 73.0 (C-2); 68.9 (C-5); 62.8 (C-6); 55.7(OCH₃.) HRMS (ESI): Calcd. For C₆H₂₃O₇ [M+H]⁺: 327.1445. found:327.1422.

4-Methoxyphenyl 2-O-allyl-3,4,6-tri-O-benzoyl-β-D-galactopyranoside(product 4)

To a solution of 3 (27 mg, 0.83 mmol) in CH₂Cl₂ (1 mL) and pyridine (65μL, 8.3 mmol), BzCl (100 μL, 8.3 mmol) was added and the reactionmixture was stirred at room temperature for 18 hours. MeOH (1 mL) wasadded and the reaction mixture was concentrated under reduced pressure.Purification with flash chromatography (1:3 EtOAc-hexanes) gave product4 (51 mg, 97%.) [α]_(D) ²⁵=+48.6° (c=0.1, CHCl₃); ¹H NMR (400 MHz, D₂O):δ8.07-7.29 (m, 15H, Ar); 7.06-6.71 (m, 4H, MeOC₆H₄); 5.89 (d, 1H, J=2.7Hz, H-4); 5.74 (m, 1H, CH₂—CH═CH₂); 5.42 (dd, 1H, J₁=3.5, J₂=10.0 Hz,H-3); 5.21-5.01 (m, 3H, CH₂—CH═CH₂, H-1); 4.57 (m, 1H, H-6a); 4.39-4.06(m, 5H, CH₂—CH═CH₂, H-6b, H-5, H-2); 3.73 (s, 3H, OCH₃.) ¹³C NMR (100MHz, CDCl₃): δ 171.2, 166.0, 165.7, 155.6, 151.2, 134.3, 133.8, 133.5,133.2, 133.1, 132.9, 130.6, 130.2, 129.8, 129.7, 129.6, 129.4, 128.8,128.5, 128.4, 118.8, 114.6 (Ar); 117.7 (CH₂—CH═CH₂); 102.8 (C-1); 78.7(C-2); 74.0 (C-3); 73.6 (CH₂—CH═CH₂); 72.2 (C-5); 69.9 (C-4); 63.5(C-6); 55.6 (CH₃.) HRMS (ESI): Calcd. For C₃₇H₃₄NaO₁₀ [M+Na]⁺: 661.2050.found: 661.2041.

4-Methoxyphenyl 3,4,6-tri-O-benzoyl-β-D-galactopyranoside (product 5)

To a solution of product 4 (45 mg, 70 μmol) dissolved in MeOH (1 mL),PdCl₂ (2 mg) was added and the reaction mixture was stirred at roomtemperature for 2 h. The reaction mixture was then filtered andconcentrated. Purification with column chromatography (1:3EtOAc-hexanes) gave product 5 (39 mg, 92%.) [α]D₂=+78.2° (c=0.1, CHCl₃);¹H NMR (400 MHz, D₂O): δ 8.08-7.28 (m, 15H, Ar); 7.07-6.72 (m, 4H,MeOC₆H₄); 5.91 (d, 1H, J=3.5 Hz, H-4); 5.45 (dd, 1H, J₁=3.5, J₂=10.1 Hz,H-3); 5.00 (d, 1H, J=7.8 Hz, H-1); 4.60 (m, 1H, H-6a); 4.44 (m, 1H,H-6b); 4.34 (m, 2H, H-5, H-2); 3.73 (s, 3H, OCH₃); ¹³C NMR (100 MHz,CDCl₃): δ 166.0, 165.5, 155.7, 150.9, 133.7, 133.4, 130.0, 129.9, 129.8,129.4, 129.2, 129.1, 128.5, 128.4, 118.6, 114.5 (Ar); 102.6 (C-1); 73.2(C-3); 71.6 (C-5); 69.7 (C-2); 68.1 (C-4); 62.3 (C-6); 55.6 (OCH₃.) HRMS(ESI): Calcd. For C₃₄H₃₀NaO₁₀ [M+Na]⁺: 621.1737. found: 621.1723.

4-Methoxyphenyl3,4,6-tri-O-benzoyl-2-O-methyl-phosphoramidyl-β-D-galactopyranoside(product 6)

To a solution of product 5 (18 mg, 0.030 mmol) and methyldichlorophosphate (70 μL, 0.30 mmol) dissolved in CH₂Cl₂ (1 mL) withmolecular sieves 4 Å, Et₃N (85 μL, 0.30 mmol) was added drop-wise. Thereaction mixture was stirred at 40° C. for 12 hours. Upon completion ofthe reaction as judged by TLC, ammonia gas was injected into thereaction mixture through a needle. After 5 min, the reaction mixture wasfiltered and concentrated. Purification with column chromatography(EtOAc) yielded product 6 (5.4 mg, 26%.) [α]_(D) ²⁵=+68.5° (c=0.05,CHCl₃); ¹H NMR (400 MHz, CHCl₃): δ8.06-7.31 (m, 30H, Ar); 7.07-6.72 (m,8H, MeOC₆H₄); 5.94 (m, 2H, H-4, H-4*); 5.54 (m, 2H, H-3, H-3*); 5.10 (m,4H, H-1, H-1*, H-2, H-2*); 4.58 (m, 2H, H-6a, H-6a*); 4.45 (m, 2H, H-6b,H-6b*); 4.35 (m, 2H, H-5, H-5*); 3.73 (s, 3H, OCH₃); 3.67 (d, 3H,³J_(PH)=11.6, POCH₃); 3.41 (d, 3H, ³J_(PH)=11.5, POCH₃*); 2.92 (d, 2H,NH₂); 2.51 (d, 2H, NH₂*.) ¹³C NMR (100 MHz, CDCl₃): δ 166.0, 165.7,165.6, 165.5, 155.8, 155.7, 150.8, 150.6, 133.8, 133.6, 133.5, 133.4,130.1, 130.0, 129.9, 129.8, 129.4, 128.9, 128.8, 128.7, 128.6, 128.5,128.4, 118.6, 114.7, 114.6 (Ar); 101.2, 101.1 (C-1); 73.9, 73.6 (C-2);72.5, 72.4 (C-3); 71.7 71.5 (C-5); 68.0 (C-4); 62.1 (C-6); 55.6 (OCH₃);53.6, 53.3 (POCH₃.) HRMS (ESI): Calcd. For C₃₅H₃₅NO₁₂P [M+H]⁺: 692.1898.found: 692.1815.

4-Methoxyphenyl 2-O-methyl-phosphoramidyl-β-D-galactopyranoside (product7)

Product 7 (2.5 mg, mmol) was dissolved in 0.25 M methanolic MeONa (1 mL)and the mixture was stirred for 1 h at room temperature before it wasneutralized with acetic acid and concentrated. Purification by flashchromatography eluting with 1:1 EtOAc-MeOH gave product 7 (1.0 mg, 73%.)

7: δ ¹H NMR (400 MHz, D₂O): δ6.97-6.83 (m, 8H, MeOC₆H₄); 5.05 (2d, 2H,H-1, H-1*); 4.28 (m, 2H, H-2, H-2); 3.91 (m, 2H, H-4, H-4*); 3.77-3.72(m, 4H, H-3, H-3*, H-5, H-5*); 3.67-3.60 (m, 10H, H-6, H-6*, OCH₃); 3.59(d, 3H, ³J_(PH)=11.5 Hz, POCH₃.) 3.56 (d, 3H, ³J_(PH)=11.5 Hz, POCH₃*.)¹³C NMR (100 MHz, CDCl₃): δ 154.5, 150.7, 117.7, 114.9 (Ar); 99.7 (C-1);77.0 (C-2); 75.3 (C-5); 71.6 (C-3); 68.6 (C-4); 60.5 (C-6); 55.6 (OCH₃);53.9 (POCH₃.)

7*: [α]_(D) ²⁵=−11.0° (c=0.01, H₂O); ¹H NMR (400 MHz, D₂O): δ6.97-6.83(m, 4H, MeOC₆H₄); 5.05 (d, 1H, J=7.8 Hz, H-1); 4.28 (m, 1H, H-2); 3.91(d, 1H, J=3.5 Hz, H-4); 3.77 (dd, 1H, J=3.5 Hz, J₂=9.8 Hz, H-3); 3.72(m, 1H, H-5); 3.67-3.60 (m, 5H, H-6, H-6′, OCH₃); 3.56 (d, 3H,³J_(PH)=11.5 Hz, POCH₃.) ¹³C NMR (100 MHz, CDCl₃): δ 154.5, 150.7,117.7, 114.9 (Ar); 99.7 (C-1); 77.0 (C-2); 75.3 (C-5); 71.6 (C-3); 68.6(C-4); 60.5 (C-6); 55.6 (OCH₃); 53.9 (POCH₃.) HRMS (ESI): Calcd. ForC₁₄H₂₃NO₉P [M+H]⁺: 380.1111. found: 380.1085.

Example 3 Immunodetection of MeOPN-6-α-D-Galp-(1→OMP andMeOPN→6-β-D-Gap-(1→O(CH₂)₅NH₂ by C. jejuni CPS Conjugate Antisera

The synthetic p-methoxyphenyl and aminopentyl glycosides of theMeOPN→6-Gal construct, compounds MeOPN→6-α-D-Gal-(1→OMP andMeOPN→6-β-D-Galp-(1→O(CH₂)₅NH₂, synthesized as described in the aboveexamples were tested for reactivity with antisera previously raisedagainst C. jejuni CPS conjugates of serotypes HS1, HS3, HS4 and HS23/36.Notably, of the listed serotypes, only HS23/36 expresses MeOPN-6-Gal.

Materials and Methods

The synthetic construct MeOPN-6-Gal was adjusted to 1 mg/ml and 2 μl wasspotted onto nitrocellulose membranes and allowed to dry. The individualspots were immunodetected with four different polyclonal antisera madeagainst different conventional conjugate vaccines in which different C.jejuni polysaccharide capsules were conjugated to CRM₁₉₇: (1) rabbitserum against an HS23/36 conjugate (final dilution 1:1000 in 20 mM Tris,pH 7.4, 0.425 M NaCl, 0.05% Tween 20 (TBST); Monteiro et al., (2009)Infect. Immun. 77, 1128-1136; U.S. Pat. No. 9,084,809); (2) rabbit serumagainst an HS4 conjugate (final dilution 1:1000; Monteiro et al., (2009)Infect. Immun. 77, 1128-1136; U.S. Pat. No. 9,084,809); (3) mouse serumagainst an HS1 conjugate (final dilution 1:500; manuscript inpreparation); and (4) mouse serum against an HS3 conjugate (finaldilution 1:500; US 2015/0273037.) Secondary antibodies used were eithergoat anti-rabbit (for HS23/36 and HS4) or goat anti-mouse (HS1 and HS3(Thermo-Pierce, Rockford, Ill.) Rabbit antibodies were obtained fromHarlan Laboratories (Indianapolis, Ind.) and mouse antibodies weregenerated in house using conventional methods. Immunoblots weredeveloped using a chemiluminesence kit (Pierce Supersignal West FemtoMaximun Sensitivity Substrate, ThermoFischer Scientific, Waltham, Mass.)and imaged on a BioRad gel imager. (Bio-Rad Laboratories, Hercules,Calif.) The conjugate with linker was analyzed using similar methods.

As illustrated in FIG. 6, the monosaccharide construct MeOPN-6-Gal wasrecognized by antibody against capsule polysaccharide isolated fromHS23/36 conjugated to CRM₁₉₇ (CPS with a MeOPN at C-6 of Gal.)Unexpectedly, antibody against polysaccharide from HS4 conjugated toCRM₁₉₇ (CPS with MeOPN at C-7 of ido-heptose) also elicited a responseequivalent to anti-HS23/36 CRM₁₉₇ conjugate against MeOPN-6-Gal. Also,anti-HS1-CRM₁₉₇ (CPS with low amounts of MeOPN at C-6 Gal) also reactedto MeOPN-6-Gal, although to a somewhat lesser extent. The HS3 CPSconjugate antisera (CPS with MeOPN at C-2 of ido-heptose) did not reactwith MeOPN-6-Gal. No reaction was observed between α-D-Gal-(1-OMP(devoid of MeOPN) and HS23/36 CPS conjugate antisera (data not shown.)Thus, the data show that antibodies generated against HS23/36, HS4 andHS1 all react with the synthetic MeOPN-6-Gal antigen. In contrast, theseantibodies do not react with heterologous capsules. In other words,there is no detectable reactivity of anti-HS23/36 antibodies withpurified HS4 or HS1 capsules.

The strong cross-reactivity with MeOPON-6-Gal exhibited against HS23/36and HS4 antibody may be explained by the fact that MeOPN-6-Gal shareepitopic structures with HS23/36 and HS4 capsule polysaccharides. Oneexplanation may be that the MeOPN group in both HS23/36 and HS4 is to aprimary hydroxyl. The cross reaction of MeOPN-6-Gal (HS23/36) with HS4,which contains MeOPN-7-6d-β-D-ido-Heptose, was unexpected, but may bedue to the linkage of MeOPN to primary hydroxyl positions on bothsugars. Indeed, as shown in FIG. 6, antibodies generated againstHS23/36, HS4 and HS1 all react with the synthetic MeOPN-6-Gal antigen.In contrast, these antibodies do not react with heterologous capsules.In other words, there is no detectable reactivity of anti-HS23/36antibodies with purified HS4 or HS1 capsules.

FIG. 7 compares the recognition of constructs MeOPN-6-α-D-Galp-(1→OMP incolumn A (same data as FIG. 6B) with data in column B using constructMeOPN→6-β-D-Galp-(1→O(CH₂)₅NH₂ using the indicated conjugate antisera.As depicted in FIG. 7, both constructs were strongly recognized byHS23/36 CPS conjugate antisera (whose CPS contains a MeOPN→6-α-D-Gallinkage in non-stoichiometric amounts), by HS4 CPS conjugate antisera(whose CPS has a non-stoichiometric MeOPN→7-6d-ido-Hep linkage), and,albeit with weaker intensity, by HS1 CPS conjugate antisera (thatcontains a very low amount of MeOPN→6-α-D-Gal.) As discussed above, thedetection of synthetic MeOPN→6-D-Gal by HS23/36, HS4, and HS1 CPSconjugate antisera points to the fact that these polyclonal preparationscontain specific antibodies for MeOPN units at primary positions. TheHS3 CPS conjugate antisera (with MeOPN at C-2 of 6d-ido-Hep in CPS) didnot react with either synthetic constructs MeOPN→6-α-D-Galp-(1→OMP orMeOPN→6-β-D-Galp-(1→O(CH₂)₅NH₂ (data not shown.) No reaction wasobserved between the Gal OMP and aminopentyl glycosides (devoid ofMeOPN) and HS23/36 CPS conjugate or whole-cell antisera (data notshown.)

As indicated in FIG. 7, within the limits of detection, no difference inantisera reactivity was observed between MeOPN→6-α-D-Galp-(1→OMP andMeOPN→6-β-D-Galp-(1→O(CH₂)₅NH₂, which suggests that the recognition ofMeOPN at the exocyclic C-6 position of Gal was not dependent on theanomeric configuration. That MeOPN→6-Gal was accessible in a conjugateformat was confirmed by the reaction of HS23/36 whole-cell sera with aMeOPN→6-Gal CRM₁₉₇ conjugate. These data indicate that the syntheticMeOPN→6-Gal entities (regardless of anomeric configuration) not onlyreact with antisera raised by homologous C. jejuni HS23/36 CPSconjugate, but also with those generated by serotypes HS1 and HS4, whichalso contain a MeOPN at a primary position (see, e.g., FIG. 1.)

Example 4 MeOPN-6-Gal is an Immunodominant Epitope

Until the discovery of a second MeOPN linkage at Gal-O-6 reportedherein, MeOPN had only been reported on the O-2 position of galactose.Kanipes et al., (2006) J Bacteriol. 188, 3273-3279. The below experimentutilizing a CPS conjugate vaccine demonstrates that the MeOPN linkage atGal-O-6 is immunodominant over MeOPN-2-Gal.

Materials and Methods

Two microliters of a 1 mg/ml solution of synthetic MeOPN-6-Gal (preparedas disclosed above) and two isomers (“A” and “B”) of MeOPN-2-Gal(prepared as disclosed herein) were spotted onto a nitrocellulosefilters using conventional methods and allowed to dry. The filters wereblocked with the blocking agent provided with Supersignal West FemtoMaximum Sensitivity Substrate (Thermo-Pierce, Rockford, Ill.) Filterswere mixed with primary rabbit polyclonal antibodies made againstformalin killed whole cells of C. jejuni strain 81-176 (final dilution1:500 in (20 mM Tris, pH7.4, 0.425 N NaCl, 0.05% Tween 20) (Bacon etal., (2001) Mol. Microbiol. 40, 769-777) or rabbit antibody to anHS23/36 polysaccharide-CRM197 conjugate vaccine (final dilution 1:1000)(Monteiro et al., (2009) Infect. Immun.77, 1128-1136.) Filters werereacted with primary antibody overnight and then washed. Secondaryantibody was goat anti-rabbit IgG (final dilution, 1:50,000)(Thermo-Pierce, Rockford, Ill.) After washing the filters were detectedwith Supersignal West Femto Maximum Sensitivity Luminescence Substrateand images were recorded on a BioRad gel imaging system (Bio-RadLaboratories, Hercules, Calif.)

As depicted in FIG. 8, results clearly indicate that the rabbit antibodyto an HS23/36 polysaccharide-CRM197 conjugate vaccine detectedMeOPN-6-Gal, but did not detect either isomer of MeOPN-2-Gal. Similarresults were obtained using the rabbit polyclonal antibodies, althoughsome reactivity was detected against MeOPN-2-Gal B isomer. These dataclearly indicate the immunogenicity of the MeOPN-6-Gal monosaccharide,and the immunodominance of the methyl phosphoramidate at the 6 positionof Gal over MeOPN at the 2 position of Gal. The immunodominance ofMeOPN-6-Gal over MeOPN-2-Gal has also been demonstrated in additionalexperiments, including studies using various mutant strains of C.jejuni, and results suggest that levels of MeOPN-6-Gal can modulate theimmune response (see Example 8 below.) These data suggest that, inaddition to the chemical synthesis of MeOPN-sugar epitopes ascontemplated herein, CPS-based vaccines against C. jejuni might beimproved by exploiting the immunodominance of MeOPN-modified sugars,e.g., by using strains that overexpress the immunodominant MeOPN-6-Galepitope for capsule purification and vaccine production.

Example 5 Conjugation of MeOPN→6-β-D-Galp-(1→O(CH₂)₅NH₂ to proteinCRM₁₉₇

The synthesis of the synthetic construct linked to a protein carrier toform a conjugate is depicted in FIG. 9 (Scheme 4.) The linker equippedgalactoside (compound 12 from FIG. 3 or compound 9 from FIG. 4) (4.5 mg)and an excess of adipic acid N-hydroxysuccinimido diester (10 equiv.)was dissolved in DMSO (1 ml.) Triethylamine (60 μl), was added drop-wiseand the reaction mixture was stirred at room temperature for 4 h. Afterconcentration under reduced pressure, the residue was extracted withH₂O, followed by purification with column chromatography (3:1EtOAc-Hexane) giving the activated monosaccharide, compound 13. Thisresulting half ester, (compound 13) was then condensed with the aminogroups of the protein CRM₁₉₇ in phosphate buffer (NaPi buffer, pH 7) toyield compound 14. Specifically, conjugation was carried out with theactivated monosaccharide with CRM₁₉₇ at a molar ratio of 100:1 (moles ofactive ester per moles of protein) in 70 mM phosphate buffer pH 7.0.After stirring 3 days at room temperature, the conjugate (compound 14)was dialyzed against running water.

The conjugation was analyzed and confirmed with SDS-PAGE gel andmALDI-TOF. Specifically, the conjugation ofMeOPN→6-β-D-Galp-(1→O(CH₂)₅NH₂ to CRM₁₉₇ was analyzed and confirmed bygel electrophoresis (FIG. 10A) Western blot (FIG. 10B) and massspectrometry (mALDI-TOF) (FIG. 10C) according to conventional methods.

Materials and Methods

The MeOPN-6-Gal construct linked to CRM₁₉₇ was analyzed andcharacterized by SDS-PAGE and immunoblotting using conventional methods.Samples of the synthetic MeOPN-6-Gal linked to CRM₁₉₇ (2.5 μg and 5 ugby weight) were analyzed on 12.5% SDS-PAGE gels and either stained withGelCode Blue (ThermoFischer Scientific, Waltham, Mass.) or transferredto nitrocellulose and immunodetected with rabbit poly-clonal antibodiesto whole cells of C. jejuni 81-176 (HS23/36) (Bacon et al., (2001) Mol.Microbiol. 40, 769-777.) The stained SDS-PAGE gel indicated that thevaccine conjugate was heterogeneous in size, ranging from slightlylarger than unconjugated CRM₁₉₇ to >250 Kd. (FIG. 10A.) Results fromimmunoblotting indicate that the vaccine conjugate reacted with rabbitpolyclonal antibodies to whole cells of C. jejuni strain 81-176indicating cross reaction between the capsule and the conjugate (datanot shown.) Due to the fact that the final product (the conjugate)contained diastereoisomers of MeOPN, only half of the MeOPN→6-D-Galpepitopes reflected those in the native CPS. Even so, Western blotanalysis with HS23/36 whole cell antisera showed that the conjugateexposed MeOPN→6-D-Gal epitopes that mimic MeOPN stereochemistry andlinkage on cell-surface (FIG. 10B.)

The conjugate was also analyzed by MALDI-TOF using conventional methodsto more accurately determine masses of the conjugate. Briefly, sinapinicacid (Sigma Aldrich, St. Louis, Mo.) was saturated in 30:70 (v/v)acetonitrile (ACN): 0.1% trifluoroacitic acid (TFA) in water as thematrix. The matrix and sample (1 mg/mL) were pre-mixed in equal volumes,and 1 L was deposited on a ground steel plate by dry droplet method foranalysis. Microflex LRT matrix-assisted laser desorption and ionizationtime-of flight (MALDI-TOF) mass spectrometer (Bruker Daltonics Inc,Billerica, Mass.) was set at linear mode with positive ion detection toobtain the mass spectra. Results indicate that the MeOPN-6-Gal-CRM₁₉₇conjugate vaccine gave a major peak of mass 61,781. The mass for CRM197in a similar MALDI experiment was 57,967 daltons (not shown.) Thus, themass difference is 3,814 daltons. Since the mass of MeOPN-6-Gal and thelinker is 461 daltons (data not shown), this indicates thatapproximately 8 MeOPN-6-Gal-linker moieties were added per CRM₁₉₇molecule. No larger form was detected, however, this may be due to thefact that larger molecules are more difficult to detect using the BrukerDaltonics instrument.

Example 6 MeOPN→*6-β-D-Gal CRM₁₉₇ Conjugate Antibodies Recognize C.jejuni HS23/36 Cell-Surface and have Bactericidal Activity

We have previously demonstrated that immunogenic capsule polysaccharideconjugate vaccines (“conventional” vaccines) against C. jejuni elicitserum bactericidal antibodies (SBAs) (manuscript in preparation.) Inother words, the antibodies generated against the conventional vaccinecan bind to the bacterium in the presence of complement and inducebacterial lysis. As discussed in the above examples, MeOPN-6-Gal hasbeen synthesized and shown to react with antibodies to conventionalCRM₁, conjugate vaccines based on both HS23/36 and HS4. A vaccineconjugate composed of MeOPN-6-Gal linked to CRM₁₉₇ with approximately 8MeOPN-6-Gal moieties per protein was synthesized as provided above andtested for immunogenicity in rabbits.

Materials and Methods

Using conventional methods and commercially available reagents, a rabbitwas immunized with four doses of MeOPN-6-Gal linked to CRM₁₉₇ vaccineconjugate (each at 250 ug) with Freund's adjuvant. The final serum wasused in an ELISA in which C. jejuni 81-176 capsule conjugated to BSA wasthe detecting antigen. The endpoint titer of the serum was 1:200. Therabbit serum generated against MeOPN-6-Gal was heat-inactivated byheating to 56° C. for 30 minutes to inactivate endogenous complement. Asa control, the pre-bleed of the same rabbit (prior to immunization) wasalso heat inactivated. Sera were serially diluted in a microtiter plate,mixed with C. jejuni 81-176 and baby rabbit complement. The plate wasincubated at 37° C. under microaerobic conditions. Aliquots from eachwell were plated onto Mueller Hinton agar plates to enumerate thesurviving bacterial cells. The results are reported as the fold-increasein killing between the pre-bleed and the final bleed of the immunizedrabbit.

The results for the rabbit immunized with the MeOPN-6-Gal-CRM₁₉₇conjugate vaccine indicated a 16-fold increase in serum bacteriocidalactivity. Results from flow cytometry are depicted in FIG. 11. Dataindicate that the conjugate vaccine (e.g., compound 14 in FIG. 9) iscapable of inducing antibodies in rabbits specific to the CPSMeOPN→6-D-Gal linkage exposed on the cell-surface of C. jejuni HS23/36cells. The intensity of binding to C. jejuni HS23/36 cells was higherusing antibodies raised by the native CPS conjugate. Intensity ofbinding to C. jejuni HS23/36 cells was lesser with the antibodies raisedto the synthetic vaccine, and a portion of the cells did not react withMeOPN→6-D-Gal antibodies at all. However, binding of these antibodies tothe surface of HS23/36 cells is consistent with the observed rise in SBAtiter discussed above.

Example 7 Synthesis of Polymeric Constructs Comprising Campylobacterjejuni Synthetic Antigens

Immunogenic synthetic constructs comprising one or more syntheticMeOPN-monosaccharides and optionally associated with one or more othersaccharides are contemplated herein. Examples of such polymericconstructs which have been synthesized are depicted herein in FIG. 15and FIG. 18.

Materials and Methods

The multi MeOPN-6-Gal polymeric conjugate of FIG. 15 was synthesizedusing conventional methods, commercially available reagents, andmonosaccharides disclosed herein and in the proceeding examples. Lintnerstarch (100 mg) was activated with 0.04 M NaIO4 in 0.1 M NaOAc buffer(100 ml) pH 4, at 4° C. for 3 days. After 2 days of dialysis againstwater, 1000 Da molecular cutoff, the product mixture was centrifuged.The supernatant was lyophilized and further purified on a Bio-Gel P-2column.

The activated starch (8 mg) was chemically conjugated withMeOPN→6-β-D-Galp-(1→O(CH₂)₅NH₂ (4 mg) in 0.1 M borate buffer (5 ml), pH9. Sodium cyanoborohydride (40 mg) was added and the reaction mixturewas stirred for 1 day at RT and 2 days at 37° C. The conjugate was thendialyzed against running water (1000 Da) for 2 days and thenlyophilized.

The starch-sugar conjugation product (4 mg) was conjugated with CRM₁₉₇(4 mg) in 0.1 M borate buffer (5 ml), pH 9. Sodium cyanoborohydride (40mg) was added and the reaction mixture was stirred for 1 day at RT and 2days at 37° C. The conjugate was then dialyzed against running water(1000 Da) for 2 days and then lyophilized. The resulting syntheticconjugate was characterized using Western gel and immunoblotting and 1HNMR as provided in FIGS. 16 and 17, respectively. Briefly, for theimmunoblot, the synthetic conjugate was electrophoresed on a 12.5%polyacrylamide gel in duplicate. Part of the gel was stained and theother part was transferred to nitrocellulose using a Transblot TurboSystem (BioRad, Hercules, Calif.) and immunodetected with rabbithyperimmune sera to formalin killed whole cells of C. jejuni strain81-176 (final dilution 1:500 in TBST which is 20 mM Tris, pH 7.4, 0.425N NaCl, 0.05% Tween 20). The filter was reacted with primary antibodyovernight and then washed. Secondary antibody was goat anti-rabbit IgG(final dilution 1:50,000 in TBST). After washing, the filter wasdetected with Supersignal West Femto Maximum Sensitivity LuminescenceSubstrate and images were recorded on a BioRad gel imaging system.

The synthetic polymeric conjugate depicted in FIG. 18 was similarlyprepared using conventional methods and reagents, and conjugated to aprotein carrier. In contrast to the conjugate depicted in FIG. 15, thesynthetic construct depicted in FIG. 18 comprises not only multipleMeOPN-6-Gal monosaccharides, but also multiple MeOPN-2-Gal andMeOPN-1-Fru monosaccharides. As described above, the variousmonosaccharides are chemically associated (conjugated) using a starchbackbone. The sugar is chemically equipped with a linker that can serveas a bridge between the sugar and the starch. A carrier protein isaffixed to the construct.

Example 8 Phase Variation of Genes Encoding Methyl PhosphoramidateTransferases in Campylobacter jejuni Modulates Capsule Structure andResistance to Complement-Mediated Killing of Campylobacter jejuni

The genes for biosynthesis of MeOPN are highly conserved among strainsof C. jejuni, while the genes encoding MeOPN transferases are highlyconserved at the 5′ end of the gene, but divergent at the 3′ end. Theconserved 5′ ends are also characterized by the presence ofhomopolymeric G tracts that undergo phase variation (PV) by slip strandmismatch repair during replication [12]. Thus, the population canconsist of mixtures of cells with these genes in either “ON” or “OFF”configurations, a trait characteristic of genes encoding multiplesurface antigens of C. jejuni [12-17]. The high frequency of PV is dueto lack of mismatch repair (MMR) enzymes [12, 18].

As discussed above, C. jejuni CPSs have been shown to be important forpathogenesis. Non-encapsulated mutants were attenuated in a ferret modelof diarrheal disease and were reduced in their ability to colonizechickens, mice and piglets [19-22], and there are conflicting reports onthe role of MeOPN in pathogenesis of a Galleria mellonella model ofdisease [22, 23].

Capsules are a major factor in resistance to complement mediatedkilling, and, in the case of C. jejuni 81-176 (HS23/36), MeOPN isessential for complement resistance. Thus, mutants expressing apolysaccharide CPS lacking MeOPN were as sensitive tocomplement-mediated killing as a mutant lacking CPS [21, 22].Polysaccharide CPSs of two serotypes, HS2 and HS23/36, have also beenshown to have immunomodulatory effects in vitro [21, 24], and theimmunomodulatory effects for 81-176 have been confirmed in vivo in amouse model [25]. MeOPN has also been shown to serve as a phage receptor[26, 27].

The 81-176 CPS is a repeating trisaccharide of galactose,3-O-methyl-6-deoxy-altro-heptose, and N-acetyl glucosamine (see FIG.19A). Strain 81-176 contains genes predicted to encode two putativeMeOPN transferases, CJJ81176_1420 and CJJ81176_1435 (FIG. 19B) [22].MeOPN has been reported on both the 2 position of galactose(MeOPN-2-Gal) and data provided herein demonstrate that an MeOPN moietyis also found on the 6 position of galactose and that anti-conjugateantibodies recognized synthetic MeOPN-6-Gal. Here, we confirm not onlythat MeOPN-6-Gal and, to a lesser extent, MeOPN-2-Gal, are theimmunodominant epitopes on intact CPS recognized by conjugate vaccines,but also that phase variation of the MeOPN epitopes can modulate CPSstructure and levels of resistance to complement-mediated killing.

Methods and Materials

Strains and growth conditions: All work was done in the 81-176 strain ofC. jejuni. Mutants of this strain are listed in Table 1.

TABLE 1 Mutants of 81-176 used in this study. Strain Strain Back-CJJ1420 CJJ1435 Refer- no. Genotype ground genotype genotype ence 3468kpsM::aph3 wildtype variable variable 34 3469 kpsM::aph3 3468 variablevariable 34 (pRY111 + kpsMT) 3390 mpnC::cat wildtype variable variable21 3391 mpnC::cat complement 3390 variable variable 21 3477CJJ1420::aph3 wildtype negative variable This work 3498 CJJ1420::aph3,3477 ON variable This hipO::CJJ1420R* + work cat 3636 CJJ1435::catwildtype variable negative This work 3637 CJJ1435::cat, 3636 variable ONastA::CJJ1435R* + aph3 3479 CJJ1420::aph3, 3477 negative negative ThisCJJ1435::cat work *R, homopolymeric tract of Gs that is subjected tophase variation was repaired.

C. jejuni was routinely cultivated on Mueller Hinton agar at 37° C.under microaerobic conditions. Media was supplemented with antibioticsas needed. For capsule extraction, cells were grown in porcine brainheart infusion broth or plates (Difco).

Oligonucleotide primers: All oligonucleotide primers used are listed inTable 2 and were synthesized by Life Technologies.

TABLE 2 Primers used in this study. Primer name Sequence SEQ ID NO.pg12.13 GGAATTCGATGATTATTTTATAGATATTG 1 GTGTGCCTGAGG pg12.14CCCTCGAGGGGATATTACTATCGACTATA 2 TCGTAACTATTACAACC pg12.25CCAGCTGAACTTGCTTGGGAGATG 3 pg12.26 GGGATATTACTATCGACTATATCGTAACT 4ATTACAACC pg10.07 GTGTGATGTGGTGGTTACGTTGAATTCGG 5 G pg10.08CTCAAATCTATAGTAAGTGGCATGATTAA 6 CATGCCAAGC pg14.67CATCCTTATCCTTCATTACTTGATCC 7 pg14.68 CGTGGAACATGTTTATTTATCATATGC 8pg12.31 CATGAAAATCCTGAGCTTGGTTTTGATG 9 pg12.32GTATTTTAAAACTAGCTTCGCATAATAAC 10 pg12.33 GCGCCCATGGGTTAACGGAGCACTTCCA 11TGACCACCTCTTCC pg12.34 GCGCCCATGGTCTAGAAGATCTCCTATTT 12ATGCTGCTTCTTTGCTTCTGG pg12.29 CGGGATCCAAAGGAGAAACCCTATGTAT 13AACCCAAACTCAGC pg12.30 GGAATTCGTAAAATCCCCTTGTTTCATAT 14TGATTCCTTTCTCTAATTTTAAACAC pg12.37 GCTATGATTGAGTTTACAAACAATGGAG 15GAGGATATATAGCATTATTTAAAAAACT C pg12.38 GAGTTTTTTAAATAATGCTATATATCCTC 16CTCCATTGTTTGTAAACTCAATCATAGC pg14.35 GGAATTCCTATATTATAAGATAATAACAC 17AATTCGCCTCCTATG pg14.03 CGGGATCCAGGAGAAACCCTATGTATAA 18 CCCAAACTCAGCpg14.09 GCTATGATTGAGTTTACAAACAATGGAG 19 GAGGATATATAGCATTATTTAAAAAACT Cpg14.10 AGTTTTTTAAATAATGCTATATACCTCCT 20 CCTTTGTTTGTAAACTCAATCATAGC

Conjugate vaccine synthesis: Capsular polysaccharide isolation andconjugation to CRM197 (Pfenex) were done as described in Monteiro et al.[30]. The three vaccines were called CCV [30], DB4, and CJCV1.

Rabbit polyclonal antisera: Rabbit hyperimmune polyclonal antibodieswere generated against three batches of HS23/36-CRM197 conjugatevaccines, CCV1 [30], DB4, and CJCV1 (Harlan Bioproducts for Science). Arabbit polyclonal serum against formalin fixed whole cells of 81-176 hasbeen reported previously [34].

PCR: All PCR products generated for cloning or sequence analysis wereamplified using Phusion high fidelity polymerase (New England Biolabs).All other PCRs used Taq polymerase (Applied Biosystems/LifeTechnologies).

Mutation of CJJ81176_1420: CJJ81176_1420 was cloned into pCRScript usingprimers pg12.13 and pg12.14 that introduced EcoR and XhoI sites,respectively. This plasmid was subjected to transposon mutagenesis usingTnp Km (kanamycin resistance or aph3; Epicentre) and individual Km^(r)transposon insertions were sequenced with primers internal to thetransposon to determine the site of insertion. A non-polar transposoninsertion at bp 367 of the 1779 bp gene was used to electroporate 81-176to Km^(r) using methods previously described. The putative mutation wasconfirmed by PCR using primers pg12.25 and pg12.26 that bracket thepoint of insertion of the kanamycin gene and this mutant was calledstrain 3477.

Mutation of CJJ81176_1435: CJJ81176_1435 was cloned into pCRScript usingprimers pg10.07 and pg10.08. The chloramphenicol resistance (cat)cassette from pRY109 [35] was cloned into a unique NcoI site located atbp 747 of the 1813 bp gene. Clones were partially sequenced to determineorientation of the cat cassette and one in which the gene was insertedin the same orientation as CJJ81176_1435 was used to electroporate81-176 to Cm^(r). Putative clones were confirmed by PCR using pg14.67and pg14.68 that bracket the NcoI site of insertion, and the resultingmutant was called strain 3636.

Construction of a double mutant in both putative MeOPN transferases:Strain 3477, CJJ81176_1420::aph3, was electroporated to Cm^(r) with thesame plasmid used to generate strain 3636, thus generating a doublemutant, strain 3479 (see Table 1).

Construction of a hipO (hippurate) insertion vector. The hipO gene of81-176 (CJJ81176_1003) was cloned into pCRScript using primer setpg12.31 and pg12.32. A unique XbaI site was introduced in the center ofthe hipO gene by inverse PCR with primer sets pg12.33 and pg12.34. Thisplasmid was called pCPE3490.

Construction of strains expressing repaired alleles of CJJ81176_1420 andCJJ81187_1435: The CJJ1420=aph3 mutant was complemented with a repairedallele as follows. The wildtype CJJ81176_1420 gene was PCR amplifiedusing primers pg12.29 and pg12.30, which introduced BamHI and EcoRIsites, respectively, and the resulting amplicon was cloned into BamHIand EcoRI digested pCPE108. Plasmid pCPE108 contains the σ28 promoterfrom flaA cloned between the XbaI and BamHI sites of pBluescript [36].The phase variable G9 tract within CJJ81176_1420 was repaired by QuickChange (Life Technologies) mutagenesis such that the G9 was changed toGGAGGAGGA using primers pg12.37 and pg12.38. The entire insert was movedas an EcoRI-NotI fragment into pBluescript and a SmaI-ended cat cassettefrom pRY109 [35] was inserted into the EcoRV site 3′ to the repairedCJJ81176_1420 gene. The entire construction (σ28-CJJ81176_1420+cat) wasPCR amplified with forward and reverse primers and cloned into theunique XbaI site within the hipO gene in pCPE3490 (described above) thathad been blunted. This construction was used to electroporate 3477, theCJJ81-176_1420::cat mutant, to Km^(r), generating strain 3498.

The CJJ1435::cat mutant was complemented by a similar strategy. PlasmidpCPE108 was modified to contain an aph3 gene at the XhoI site in thepolylinker, generating pCPE3583. CJJ81176_1435 was PCR amplified usingprimers pg14.35 and pg14.03, which introduced BamHI and EcoRI sites,respectively, and cloned into BamHI and EcoR1 digested pCPE3583. Thephase variable G9 tract located within the coding region ofCJJ81176_1435 was subjected to site-directed mutagenesis as describedabove using primers pg14.09 and pg14.10. The repaired CJJ81176_1435 geneand the adjacent aph3 gene were PCR amplified using forward and reverseprimers and cloned into an EcoRV site on a plasmid containing the astAof strain 81-176, as previously described [36, 37]. This plasmid wasused to electroporate the CJJ81176_1435 mutant, 3636, to Km, generatingstrain 3637.

CPS immunoblots: Whole cells of C. jejuni were digested with proteinaseK as previously described [30, 34]. Preparations were electrophoresed on16% Tris-glycine gels (Invitrogen) and stained with silver (BioRad) tovisualize the LOS cores. Equivalent core amounts were electrophoresed on12.5% SDS-PAGE gels and transferred to nitrocellulose. The whole cellanti-81-176 serum was used at a final dilution of 1:500, and theanti-conjugate antisera were used at a final dilution of 1:100.Immunoblots were developed using chemiluminescence (SuperSignal WestFemto Maximum Sensitivity Substrate, ThermoFisher) and recorded on aBioRad Gel Imaging System.

NMR analyses: ³¹P NMR spectra were recorded using a Bruker AMX 400spectrometer. 1D 31P and ¹H-³¹P 2D experiments were performed usingBruker software. Samples were lyophilized with D2O (99.9%) three timesprior to recording the spectra. Orthophosphoric acid (δ_(P) 0.0) wasused as the external reference for all ³¹P experiments.

Anti-CPS ELISAs: To determine the anti-CPS response in hyperimmunerabbits Carbo-BIND plates (Corning, Corning, N.Y.) were coated with 100μl of oxidized CPS from wildtype, 3390, 3477 or 3636 strains (2 g/ml insodium acetate buffer (pH 5.5) for 1 hr at room temperature according tothe manufacturer's instructions. Plates were washed with 1×PBS-0.05%Tween-20 (PBST), blocked with 5% fetal calf sera in PBST (5% FCS-PBST)for 1 hr at 37° C. and washed again with PBST. All rabbit hyperimmunesera were serially diluted in 5% FCS-PBST in duplicate and incubated for1.5 hr at 37° C. After washing, HRP-conjugated goat anti-rabbit IgG(Sigma-Aldrich, St. Louis, Mo.) was diluted in 5% FCS-PBST and added at100 μl per well for 1 hr at 37° C. before washing.2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS)-peroxidasesubstrate (KPL, Gaithersburg, Md.) was used as a detection reagent andthe OD405 was measured. The mean OD405 of negative control wells(coating buffer alone)+3 standard deviations was used to determine theendpoint titer.

To determine the levels of MeOPN-6-Gal on the three CPS-CRM197conjugates, the conjugates were normalized based on total CPS contentand serially diluted on MaxiSorp Nunc® plates (Sigma-Aldrich, St. Louis,Mo.) in carbonate coating buffer overnight at 4° C. Plates were washedwith PBST and blocked with BSA in PBST for 1 hr at 37° C. To detectMeOPN-6-Gal, plates were washed and DB3 monoclonal was diluted inblocking buffer and incubated for 1 hr at 37° C. Goat anti-mouse IgG-HRP(Thermo-Scientific) was added after washing and incubated for 1 hr at37° C. Plates were washed and 100 μl of tetramethylene benzidine (TMB,eBioscience, San Diego, Calif.) substrate was added for 10 min before100 μl 1M H₂SO₄ was added to stop the reaction. The OD was read at 450nm.

Generation of hybridomas: Splenocytes from BALB/c mice immunizedsubcutaneously with CPS81-176-CRM₁₉₇ conjugate three times at 4 weekintervals, were fused with SP2/O myeloma cells to generate hybridomasaccording to [38]. Briefly, splenocytes and SP2/O cells were fused inthe presence of polyethylene glycol and mixed with peritonealmacrophages derived from a non-immunized BALB/c mouse in hybridoma media[Iscoves media containing 20% FBS, 2×HAT (200 mM hypoxanthine, 0.8 mMaminopterin, 32 mM thymine), OPI (1 mM oxaloacetate, 0.45 mM pyruvateand 0.2 U/mL insulin), 4 mM glutamine and IL-6 (10 ng/ml). Fused cellswere immediately plated on eight 96-well cell culture plates andincubated at 37° C. in a 5% CO2 atmosphere for 2 weeks. Hybridomas wereselected by screening culture supernatants from each well by ELISA usingBSA conjugates of CPS from both 81-176 and the mpnC mutant as antigenictargets.

Production and purification of monoclonal (mAb) DB3: A single cellhybridoma clone was gradually expanded into 16 T-150 flasks, whileweaning down to 2.5% FBS in Iscove's media. Cells were transferred into2 L roller bottles containing 1 L of serum free media (SFM), and werecultured at 37° C. in 5% CO₂ for 4 weeks. The mAb DB3 in SFM waspurified over a MEP-HyperCel column according to manufacturer'sinstructions (Pall life Sciences). Eluted antibodies were dialyzed intoTBS (0.05 M Tris/0.15 NaCl, pH 7.6), and protein content was determinedby BCA assay. Aliquots were stored at −80° C. for furthercharacterization and use. Isotype was determined using an isotyping kit(Pierce).

Dot blot assay: C. jejuni cells were set to an OD₆₀₀=0.5 in PBS, pH 7.4,and spotted in triplicate (2 microliters) onto nitrocellulose andallowed to dry. Membranes were immunodetected using mAb DB3 at a finalconcentration of 10 ug/ml, followed by anti-rabbit goat IgG-HRP(Sigma-Aldrich) and then chemiluminescence detection, as describedabove.

Complement killing: Pooled normal human sera (NHS) was purchased fromSigma and a single lot was used for all experiments. Assays were done asdescribed in Maue et al. [21], except that a range of NHS was used.Assays were repeated between 2-9 times for each strain. Statistics weredone using Graphpad Prism.

Colony blots with anti-conjugate antibody reactivity: Individualcolonies of 81-176 were grown for 48 h on MH agar plates. Individualcolonies were resuspended in PBS and 2 ul was spotted ontonitrocellulose for immunodetection with DB3 antibody at a final dilutionof 10 μg/mL.

Flow cytometry: 81-176 strains were grown for 20 hours on MH agar, andthe cells were harvested into 5 mL of PBS and filtered through a 1.2micron filter. The resulting suspension was adjusted to an OD₆₀₀ 0.1,and one ml was spun down at 12000 g for 2 min. Pellets were resuspendedin 0.5 ml 4% formaldehyde and incubated on a rotator for 10 min at roomtemperature. Cells were centrifuged, washed twice in ice-cold PBST, andresuspended in 100 microliters of a 1:50 dilution of serum fromhyperimmune rabbits immunized with a conjugate antibody or DB3monoclonal antibody at a final concentration of 112 μg/ml and incubatedfor 30 minutes at 4° C. Suspensions were washed twice with ice cold PBSTand then incubated with donkey anti-rabbit IgG AlexaFluor 647(Biolegend, San Diego, Calif.) for the hyperimmune sera or ratanti-mouse IgG1 PE (SouthernBiotech, Birmingham, Ala.), and incubatedfor 30 minutes at 4° C. The suspensions were washed twice in ice coldPBST and resuspended in 0.5 ml PBST and read on a Canto FACS. Data wereanalyzed using FlowJo (TreeStar, Ashland, Oreg.).

Vaccines DB4 and CJCV1 were produced by Dalton Pharma, Toronto.

Results

MeOPN is an immunodominant capsular epitope recognized by anti-conjugateantibodies. Proteinase K digested whole cells of C. jejuni 81-176 andmutants were immunodetected with rabbit polyclonal antibodies toformalin killed whole cells of 81-176 (FIG. 20A, [34]) and to an81-176-CRM197 conjugate vaccine, CJCV1 (FIG. 20B). FIG. 20A shows thatthe whole cell antiserum reacted with the wildtype capsule, but not anunencapsulated mutant, kpsM, strain 3468 [34]. When kpsM wascomplemented in trans (strain 3469) [34], reactivity was restored. FIG.20A also shows that the whole cell serum reacted with the mpnC mutant(strain 3390) that expresses CPS lacking MeOPN, and its complement,strain 3391 [21]. In comparison, the rabbit polyclonal serum to the CPSconjugate vaccine, (CJCV1; FIG. 2B) reacted with wildtype 81-176, butdid not react with either the CPS-void kpsM mutant or the MeOPN-voidmpnC mutant. Reactivity was restored when both mutants were complemented(strains 3469 and 3391, respectively). Similar reactivity was seen withrabbit polyclonal antisera to two other batches of 81-176-CRM197conjugate vaccines.

Reactivity of anti-conjugate antiserum to mutants of each MeOPNtransferase. Strain 81-176 contains two putative MeOPN transferasegenes, CJJ81176_1420 and CJJ81176_1435 (FIG. 19B). We generatedmutations in each gene as described in Materials and Methods, and wecompared the ability of antibodies generated to 81-176-CRM₁₉₇ conjugatevaccines to react with mutants in each of the MeOPN transferase genes.FIG. 20A shows that the antiserum to whole cells of 81-176 reacted withproteinase K digested whole cells of both putative transferase mutants,3477 and 3636, confirming expression of polysaccharide CPS. There was adistinct difference between the reactivities of the anti-whole cellserum and the anti-conjugate serum against the CPS preparation fromstrain 3477, the CJJ81176_1420 mutant. In the case of the anti-conjugateserum (FIG. 20B), a reaction could only be seen with CPS material ofapparent low mass, whereas with whole-cell sera (FIG. 20A) a broaderreactivity was observed. The intensity and pattern of the reaction withanti-conjugate sera was restored in the complement, 3498 (FIG. 20B). Theantiserum against whole cells reacted with CPS from 3636 (FIG. 20A) andthe complement. However, there was no detectable reaction between theanti-conjugate serum and CPS from 3636, the mutant in CJJ81176_1435(FIG. 20B). Reactivity with anti-conjugate serum was restored in thecomplement, strain 3637 (FIG. 20B).

Similar results were observed with additional rabbit antisera to twoother batches of conjugate vaccines. Collectively, these data indicatedthat while antibodies generated against formalin-killed whole cells of81-176 reacted with the polysaccharide chain, antibodies generatedagainst the conjugates were directed primarily to MeOPN-6-Gal and to alesser extent MeOPN-2-Gal.

The immunodominance of MeOPN in the conjugates was also examined byELISA. Anti-CJCV1 antibodies were serially diluted and reacted to CPSfrom wildtype 81-176 or mutants. The results, shown in FIG. 20C,indicated that the reaction of CJCV1 was strongest to the wildtype CPS(titer: 5.9×10⁶). There was a reaction to CPS purified from 3477, themutant in CJJ81176_1420, with an endpoint titer lower than wildtype(titer: 6.6×10⁵). Reaction of CJCV1 sera was reduced to the CPS fromboth 3636, the mutant in CJJ81176_1435 and to 3390 (mpnC) that lacks allMeOPN (titers: 600 and 8100, respectively). Based on the strongerreactivity of anti-conjugate antibodies to synthetic MeOPN-6-Gal than toMeOPN-2-Gal reported herein, it is likely that the CJJ81176_1435transferase is responsible for addition of MeOPN to the 6 position ofGal and that the transferase encoded by CJJ81176_1420 is responsible foraddition of MeOPN to the 2-position of Gal. This was further studied byNMR, below.

MeOPN modifications on the 81-176 CPS. Previously, using massspectrometry we detected a non-stoichiometric MeOPN unit at the 2position of galactose (MeOPN-2-Gal) in 81-176 CPS ([31]), with a ³¹Presonance similar to that in FIG. 21A (peak Y). Here, we confirmed thisMeOPN-2-Gal linkage by NMR (FIG. 22A) through the detection of across-peak between the ³¹P resonance Y (δ_(P) 14.45) of MeOPN and H-2(δ_(H) 4.52) of the galactose unit in a ¹H-³¹P correlation experiment.

In some 81-176 CPS preparations, albeit of lower intensity, the ³¹P NMRspectrum displayed an additional resonance (FIG. 21B) at δ_(P) 14.15(designated peak Z). A similar peak was also observed in the 31P NMR ofmutant in CJJ81176_1420, called 3477, which exhibited a cross-peak (FIG.22B) between the phosphorous of MeOPN and H-6 resonances of some of theCPS galactose units, which resonated very near the methyl resonances ofMeOPN (δ_(H) 3.75 to 3.81). No significant peak Y was observed in CPSfrom 3477. The NMR data suggested that peak Z in 81-176 and 3477 (themutant in CJJ81176_1420) corresponded to a non-stoichiometric placementof MeOPN at position 6 of galactose (MeOPN-6-Gal), consistent with thedata using synthetic MeOPN-6-Gal described herein.

The ³¹P NMR spectrum of 3636 (FIG. 21C), mutant in CJJ81176_1435, didnot show either peak Y or peak Z, but yielded a previously unseenphosphorous resonance at δ_(P) 14.79 (designated peak X). A 2D ¹H-³¹PNMR experiment showed a connection between peak X and a proton resonanceat δ_(H) 4.85 (FIG. 22C). The NMR data of the CPS from strain 3636implied a new activity in the mutant in CJJ81176_1435 that afforded aMeOPN modification not previously observed in 81-176 CPS. This new CPSmodification is the subject of an on-going study.

The ³¹P NMR spectrum of a mutant in both transferases (strain 3479)showed no MeOPN-related resonances (data not shown).

Monoclonal DB3. A mouse monoclonal antibody (isotype IgG1) was isolatedfrom an animal immunized with an 81-176-CRM197 conjugate vaccine. Themonoclonal was used in a dot blot with whole cells of wildtype 81-176and various mutants, as shown in FIG. 23A. The monoclonal did not reactwith either the mpnC mutant (3390) or the 81176_CJJ1435 mutant (3636),but reactivity was restored when both mutants were complemented (3391and 3637, respectively). Since DB3 bound to the 81176_CJJ1420 mutant(3477), the specificity of the monoclonal was for the site modified bythe transferase encoded by CJJ81176_1435, which is likely MeOPN-6-Gal.Additionally, DB3 failed to react to other MeOPN-containing capsules(HS1, HS2, HS3, HS4, and HS15) [39-44], again confirming the specificitywas not to MeOPN alone, but included the sugar linkage (data not shown).

Flow cytometry analyses using DB3. FIG. 23B shows that monoclonal DB3bound to the surface of wildtype 81-176 as measured by flow cytometry,but did not bind to the mpnC mutant, as expected from the dot blottingstudies. Binding was partially restored in strain 3391, the complementof the mpnC mutant. Similarly, DB3 did not bind to 3636, the mutantpresumably lacking MeOPN-6-Gal, and binding was partially restored in3637, the complement (FIG. 23 C). However, DB3 binding to 3477, themutant lacking MeOPN-2-Gal, but retaining MeOPN-6-Gal, was reduced.Binding was enhanced in strain 3498, the complement (FIG. 23D).

Levels of MeOPN-6-Gal on conjugate vaccines modulate the immuneresponse. When DB3 was used in an ELISA to measure the levels ofMeOPN-6-Gal on three independently produced conjugate vaccines,differences in binding could be detected (FIG. 24A). CCV, the vaccineshown to protect non-human primates against diarrheal disease [30],showed the highest binding, DB4 was intermediate, and CJCV1 showed thelowest. Endpoint titers were determined by ELISA to capsules purifiedfrom wildtype 81-176 and the mpnC mutant for rabbit hyperimmune antiseraagainst each of the three vaccines, as shown in FIG. 24B. Each vaccineelicited high titers of antibodies to the intact wildtype capsule (CCV:6.6×10⁵, DB4: 4.0×10⁶, CJCV1: 5.9×10⁶), but the titers against the mpnCcapsule increased as the amount of MeOPN-6-Gal on each vaccine decreased(CCV: 100, DB4: 5400, CJCV1: 8100). Thus, the anti-polysaccharideresponse was lowest for CCV, intermediate for DB4 and highest for CJCV1.FIG. 24C-E shows the reactivity of each rabbit hyperimmune sera to thesurface of wildtype and the mpnC mutant. CCV, with the highest amount ofMeOPN-6-Gal, bound to the surface of wildtype 81-176 and no binding wasdetected to the mpnC mutant, 3390 (FIG. 24 C). Binding was enhanced inthe complement, strain 3391. Antibodies to conjugate DB4 bound to thesurface of wildtype 81-176 and showed enhanced binding to the mpnCmutant compared to CCV (FIG. 24D). Finally, antibodies to CJCV1 boundequally well to wildtype and the mpnC mutant (FIG. 24E). None of theantibodies bound to the kpsM mutant. Thus, surface binding to the mpnCmutant was enhanced as the levels of MeOPN-6-Gal were reduced in thevaccines.

Role of MeOPN in complement resistance. Van Alphen et al. [22, 31]constructed a double mutant in both putative transferase genes andshowed that the resulting mutant was sensitive to complement killing,consistent with previous work using the mpnC mutant [21, 22]. Wecompared serum resistance of 3477, the mutant in CJJ81176_1420, and3636, the mutant in CJJ81176_1435, and a double mutant lacking bothtransferases, 3479 (see Table 1) using increasing amounts of NHS. Theresults, shown in FIG. 25, indicated that at all concentrations of sera,the CJJ81176_1435 mutant, 3636, was significantly more resistant thanwildtype, and that the CJJ81176_1420 mutant, 3477, was significantlymore sensitive than wildtype 81-176 at concentrations of NHS rangingfrom 5-15%. When both mutants were complemented with repaired alleles,the levels of serum resistance returned to levels that were notsignificantly different to that of wildtype. However, mutation of bothMeOPN transferases (strain 3479) resulted in enhanced sensitivity overthe CJJ81176_1420 mutant (3477), and showed levels of sensitivitysimilar to that reported previously for another double transferasemutant [22] and for the mpnC mutant [21].

Phase variation of MeOPN-6-Gal. Strain 81-176 was plated for singlecolonies on MH agar and 60 individual colonies were dot blotted with theDB3 monoclonal antibody to measure levels of expression of MeOPN-6-Gal.The results indicated that there was considerable heterogeneity inexpression of MeOPN-6-Gal within the population, as shown by therepresentative colonies in FIG. 26A. Colonies were scored subjectivelyfor intensity, as shown in the figure. Interestingly, the population,labeled as “WT” in FIG. 26A, bound less antibody than most of the singlecolonies, which is a reflection of the heterogeneity of the population.Collectively, as shown in FIG. 26B, 54% of the single colonies werescored as “3+”, 16% as “2+”, 26% as “1+” and 4% were negative.

Discussion

Approximately 75% of C. jejuni strains contain genes encoding MeOPN intheir CPS locus, but the sugar modified and the sites of modificationhave been determined in a limited number of CPS types. Most strainscontain a single MeOPN transferase, but some, including NCTC 11168 and81-176, contain two transferases and both capsules are modified at twosites [31, 40,] and as disclosed herein. In the presence of both MeOPNtransferases in wildtype 81-176, MeOPN is attached to both the 2 and 6positions of Gal. When CJJ81176_1420 was mutated, MeOPN was attachedonly to the 6 position, presumably by the MeOPN transferase encoded byCJJ81176_1435. This also indicates that the transferase encoded byCJJ81176_1420 is responsible for addition of MeOPN to 2-Gal. However,when CJJ81176_1435 was mutated, no MeOPN was detected at either the 2 or6 position of Gal, and there was a new site of attachment at an unknownposition. This attachment must be mediated by the transferase encoded byCJJ81176_1420, since the new attachment (peak X in FIG. 21C) was notobserved in the double MeOPN transferase mutant 3479. This suggests thatthe enzyme encoded by CJJ81176_1420 has a relaxed specificity that isdependent on the secondary structure of the polysaccharide chain.Additional studies on the specificities of MeOPN transferases areongoing in our laboratories.

The MeOPN modifications on the 81-176 CPS are immunodominant inconjugate vaccines, and the response to MeOPN-6-Gal was stronger thanthat to MeOPN-2-Gal. This is also consistent with the observedreactivity of anti-conjugate antibodies with synthetic MeOPN-6-Galdescribed herein. The immunodominance of MeOPN-6-Gal may be due tohigher levels of this modification on the CPS or it may be thatmodification on the 6-position of Gal, which is on a primary hydroxyl,is more immunogenic. No antibodies to the polysaccharide chain of thempnC mutant can be detected by immunoblotting of crude CPS preparationsusing any of the rabbit hyperimmune sera to conjugate vaccines, althoughvarying levels of anti-polysaccharide antibodies could be detected bythe more sensitive techniques of ELISA and flow cytometry. However, thelevels of reactivity to the polysaccharide chain increased as the levelsof MeOPN-6-Gal decreased, as measured by DB3 binding. In contrast to theresponse to conjugate vaccines, rabbit hyperimmune serum made againstformalin-killed whole cells of 81-176 reacted with the mpnCpolysaccharide by immunoblotting. It may be that the cells used asantigen to generate this antiserum expressed less MeOPN than otherpreparations, or that some MeOPN was lost upon formalin treatment, andthus the cells contained more exposed polysaccharide.

Monoclonal DB3 appears to be specific for the MeOPN-6-Gal epitope asdetermined by whole cell dot blot, and, consistent with this, bound tothe surface of wildtype 81-176, but not to the CJJ81176_1435 or mpnCmutants by flow cytometry. Interestingly, surface binding of DB3 wasdisrupted by mutation of CJJ81176_1420, suggesting that loss ofMeOPN-2-Gal alters the secondary and/or tertiary structure of the CPSand reduces accessibility of DB3 to the surface of the cell. Although nostudies have been reported, it is likely that the polysaccharide chainis decorated with MeOPN as it is being synthesized in the cytoplasm.Decoration of sugars with MeOPN is likely to affect changes in foldingof the polysaccharide, which, after assembly on the cell surface, couldalso affect interactions between adjacent polysaccharide chains, thusaffecting accessibility of the polysaccharide to antibodies and/orcomponents of the complement cascade. This is consistent with ourobservations that loss of MeOPN-2-Gal in the CJJ81176_1420 mutantresulted in a significant reduction in resistance to complement mediatedkilling.

In contrast to the mutation of CJJ81176_1420, mutation of CJJ81176_1435resulted in enhanced resistance to complement-mediated killing. The³¹P-NMR studies indicated that the CJJ81176_1435 mutant lost bothMeOPN-2-Gal and MeOPN-6-Gal and had gained a new MeOPN modification atan undetermined site. Presumably, this new site of modification providesenhanced protection from the complement cascade. This hypothesis isconsistent with the lower endpoint titer seen for the CJJ81176_1435mutant (3636) compared to the mpnC mutant. Thus, the new MeOPNmodification may block accessibility of the anti-CJCV1 antibody to thepolysaccharide chain.

Additional studies will be needed to understand the mechanism of thesedifferential sensitivities to complement-mediated killing. However,immunoblotting of individual colonies of wildtype 81-176 with the DB3monoclonal revealed extensive heterogeneity of MeOPN-6-Gal levels withinthe population and this, in turn, would suggest that the population canreadily adapt to changing environments. Similarly, MeOPN has been shownto be required for binding of several phages, and the ability to varylevels of MeOPN could be critical for survival in several environments[26, 27]. Thus, PV appears to provide a novel mechanism to adjust tothese environmental changes, and perhaps to others encountered by thiszoonotic pathogen. Similar heterogeneity has been observed not only insurface antigens of C. jejuni [12-17, 34, 46], but also in purinebiosynthetic genes that affect the stress response [47, 48].Collectively, these data support the notion that C. jejuni is aquasi-species that survives by selection of a subset of pre-existingvariants within a heterogenous population [47].

The immunodominance of MeOPN in conjugate vaccines appears to becomparable to the immunodominance of O-acetyl groups on thepolysaccharide conjugates based on other bacterial pathogens [49-51].Importantly, there was no detectable reaction of the anti-conjugate serawith CPSs from other serotypes that express MeOPN on different sugars(data not shown), suggesting that the reaction is specific to the Gallinkages found in 81-176, and not to MeOPN per se. Non-stoichiometricmodifications to sugars confer considerable heterogeneity topolysaccharide chains and can affect immunogenicity [40, 52]. These datasuggest that CPS-based vaccines against C. jejuni might be improved byexploiting this immunodominance of MeOPN-modified sugars. It iscontemplated herein that one approach would be to use strains thatoverexpress the immunodominant epitope for capsule purification andvaccine production. Another alternative approach would be chemicalsynthesis of the MeOPN-sugar epitopes and conjugation to a carrierprotein such as contemplated herein.

Complement mediated killing of C. jejuni has been reported to occurprimarily by the classical pathway [22, 53], and the CPS likelyfunctions to shield the cell from naturally occurring antibodies in NHSthat cross-react with surface proteins. However, the MeOPN decorationsmay shield surface proteins and the polysaccharide from cross-reactiveantibodies. This is consistent with an earlier observation that NHScontains low levels of antibodies that cross-react with surface proteinsof C. jejuni and could induce low levels of complement mediated killingof multiple strains, but that, within 48 h of infection with C. jejuni,patients developed higher-level serum bactericidal titers that werestrain specific [53], an observation that may relate to CPS-specificantibody responses. Conjugate vaccines against several othergram-negative pathogens induce bactericidal antibodies that correlatewith protection [54-56], and we are exploring this possibility for C.jejuni conjugate vaccines. Although C. jejuni is generally considered tobe relatively serum sensitive [57], we have shown here that the organismhas the ability to modulate its levels of resistance via PV of the genesencoding the MeOPN transferases, and that the population is composed ofcells expressing various levels of these modifications. Thus, the levelsof serum resistance measure in vitro for a population may not reflectthe levels of resistance that can be achieved in vivo.

Thus, based on the foregoing data, we have demonstrated that thepolysaccharide capsule of Campylobacter jejuni strain 81-176 isdecorated non-stoichiometrically with methyl phosphoramidate (MeOPN) atthe 2 (MeOPN-2-Gal) and 6 positions of galactose (MeOPN-6-Gal), andthese are the immunodominant epitopes recognized by antibodies to 81-176capsule conjugate vaccines. A mouse monoclonal antibody specific forMeOPN-6-Gal bound to the surface of wildtype 81-176, but binding to amutant lacking MeOPN-2-Gal was reduced, suggesting that loss ofMeOPN-2-Gal effects changes in capsule secondary/tertiary structure.Using the MeOPN-6-Gal-specific monoclonal antibody we have shown thatthe population consists of a heterogenous mixture of cells expressingdifferent levels of MeOPN-6-Gal as a result of phase variation of theMeOPN transferase. A mutant in the MeOPN transferase encoded byCJJ81176_1420, which appears to be responsible for attachment ofMeOPN-2-Gal, was significantly more sensitive to complement-mediatedkilling than wildtype at 5, 10 and 15% normal human serum (NHS). Incontrast, the mutant in CJJ81176_1435, the transferase that appearsresponsible for addition of MeOPN-6-Gal, was significantly moreresistant than wildtype at 5, 10, 15 and 20% NHS. In the CJJ81176_1435mutant, both MeOPN-6-Gal and MeOPN-2-Gal were lost and a new,unidentified site of MeOPN modification was observed that was likelyresponsible for the enhanced serum resistance in this mutant. Thus, itappears that in the absence of the transferase encoded by CJJ81176_1435,the CJJ81176_1420 transferase was able to modify a secondary site on thecapsule. Thus, phase variation of the MeOPN transferases modulates thestructure of the capsule and the levels of resistance tocomplement-mediated killing.

REFERENCES FOR EXAMPLE 8

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Example 9 Prophetic Method of Inducing an Immune Response Against C.jejuni in a Subject

The immunogenic synthetic constructs described in the proceedingexamples can be included in an immunogenic formulation (e.g., a vaccineformulation) against C. jejuni and administered to a subject forinducing an immune response against C. jejuni. Thus, the present exampleis a prophetic method for the induction of an immune response to C.jejuni in a subject, and particularly, a method of inducing an immuneresponse in a subject that provides protective immunity from thegastrointestinal and other debilitating effects associated withcampylobacter enteritis, is contemplated herein.

As an example, such method could comprise administering an immunogeniccomposition comprising one or more synthetic constructs of the instantinvention, wherein the construct is optionally conjugated to a carriermolecule, preferably to a carrier protein molecule such as CRM₁₉₇. Themethod may further comprise one or more subsequent steps comprisingadministering one or more boosting doses of a composition comprising thesame immunogen administered in the first step.

As understood by one of skill in the art, optimal methods for inducingprotective immunity in humans are preceded by studies in animals such asin mice and monkeys. For each vaccine formulation comprising a syntheticconstruct of the instant invention, a limited amount of experimentationis required to ascertain the optimal effective dose ranges. For example,in one embodiment, it is contemplated herein that the range of a unitdose of immunogenic synthetic construct may be from about 0.1 μg to 10mg per dose in a range of buffer solutions. Optionally, subsequent to apriming dose, one or more, e.g., 2 to 4 boosting doses may also beadministered with a unit dose range of from about 0.1 μg to 10 mg ofimmunogen in a buffered aqueous solution.

Thus, a method of inducing an immune response in a subject against C.jejuni may comprise the steps of: (a.) administering an immunogeniccomposition comprising one or more synthetic constructs of the instantinvention, wherein the construct is conjugated to a carrier molecule,preferably to a carrier protein molecule, and the compositionadministered at a dose range of 0.1 μg to 10 mg per dose with or withoutan adjuvant; and (b) optionally administering a boosting dose of thecomposition as described in step (a), with or without adjuvant, at adose range of 0.1 μg to 10 mg per dose.

It is contemplated herein that depending on the route of administration,the vaccine formulation can be administered with or without any of anumber of adjuvants such as those described in detail above.

Moreover, as discussed hereinabove, the method may be performed using asynthetic construct that is conjugated to a carrier protein or using anunconjugated synthetic construct. The method may comprise the use of anyof a number of carrier molecules discussed above. As an example, CRM₁₉₇can be used. ETEC proteins may also be used as carrier proteins asdiscussed above, e.g., as disclosed in US 2015/0258201 A1.

The construct:carrier protein ratio (w/w) may be 1:1, or may be suchthat more than one construct is linked to a single carrier protein,e.g., from 2:1 to 10:1 or more; particularly, at least 8:1. As one ofskill in the art will appreciate, a single carrier molecule may beconjugated to a large number of synthetic constructs, e.g., hundreds oreven thousands of constructs per carrier molecule. An appropriate ratiobest suited to inducing and/or enhancing an immune response in a subjectmay be discerned by one of skill in the art without undueexperimentation.

Indeed, as contemplated herein, one of skill in the art could optimizethe immunogenicity of a synthetic construct for use in the methods ofthe instant invention by using different combinations of syntheticconstructs, including constructs and conjugates comprising more than oneMeOPN modified monosaccharide, adjuvants, carrier proteins, additionalimmunoregulatory agents, and routes of administration. For example, itis contemplated herein that different ETEC proteins may be used invarious combinations with the immunogenic synthetic constructs of theinstant invention to produce a construct with enhanced immunogenicity,not only to C. jejuni but also to other bacterial pathogens. To thisend, the teachings of US2015/0258201 A1 are incorporated by referenceherein in its entirety. Moreover, a composition of the instantinvention, e.g., pharmaceutical formulations, and particularly vaccineformulations of the instant invention can be administered orally,nasally, subcutaneously, intradermally, transdermally, transcutaneouslyintramuscularly, or rectally. Methods of administration and dosingregimens best suited to producing an immune response in a subject may bediscerned by one of skill in the art using conventional methods andwithout undue experimentation.

Example 10 Multivalent Vaccine Formulations Against C. jejuni or OtherOrganisms

Data provided herein demonstrate that antibodies to HS23/36, HS4 and HS1strains of C. jejuni can react with a synthetic MeOPN-6-Gal construct.Thus, in one embodiment, it is contemplated herein that one of skill inthe art, using conventional methods and without undue experimentation,could develop a multivalent vaccine formulation comprising the syntheticMeOPN-6 Gal construct disclosed herein which should cover at least thesethree major capsule types of C. jejuni.

It is further contemplated herein that additional multivalentformulations comprising one or more immunogenic synthetic constructs ofthe instant invention could be developed which cover the strains of C.jejuni which account for a majority of worldwide cases ofcampylobacteriosis. Such formulations might be produced, for example, bysynthesizing additional constructs comprising capsular monosaccharidesfrom C. jejuni strains of relevance in this regard and testing suchsynthetic constructs for immunogenicity (including possible crossreactivity) against such strains of C. jejuni. In a particularembodiment, such synthetic constructs may comprise one or moremonosaccharides comprising one or more MeOPN moieties including, e.g.,one or more MeOPN-6-Gal moieties and/or one or more MeOPN-2-Galmoieties. A synthetic construct comprising MeOPN-2-Gal is contemplatedherein.

A multivalent vaccine formulation of the instant invention may comprisea single synthetic construct designed to cover more than one strain ofC. jejuni, and/or may comprise a synthetic construct designedspecifically against a single particular strain of C. jejuni. Inaddition, one of skill in the art will appreciate that syntheticconstructs may be produced which are immunogenic not only against morethan one strain of C. jejuni, but also against more than one type ofbacterium, e.g., ETEC or Shigella, by chemically linking variousdifferent antigenic components against these additional bacteria to animmunogenic construct against C. jejuni. See, e.g., US 2015/0258201.

Having described the invention, one of skill in the art will appreciatethat many modifications and variations of the present invention arepossible in light of the above teachings. It is therefore, to beunderstood that, within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

What is claimed is:
 1. An immunogenic synthetic construct capable ofinducing an immune response against Campylobacter jejuni (C. jejuni) ina subject, wherein said immunogenic synthetic construct comprises one ormore monosaccharides comprising one or more O-methyl phosphoramidate(MeOPN) moieties, wherein said one or more MeOPN moieties is one or moreMeOPN→6 Gal monosaccharides, and wherein said immunogenic syntheticconstruct is conjugated to a carrier protein, wherein said carrierprotein contains at least one T-cell epitope.
 2. The immunogenicsynthetic construct of claim 1 wherein the carrier protein is CRM₁₉₇. 3.The immunogenic synthetic construct of claim 1 wherein the subject is ahuman.
 4. A composition comprising the immunogenic synthetic constructof claim
 1. 5. The composition of claim 4 wherein the composition is apharmaceutical composition.
 6. The pharmaceutical composition of claim 5wherein said pharmaceutical composition is a vaccine formulation.
 7. Thevaccine formulation of claim 6 wherein the formulation further comprisesone or more adjuvants.
 8. The vaccine formulation of claim 7 wherein theadjuvant is selected from the group consisting of toll-like receptorligands, aluminum phosphate, aluminum hydroxide, monophosphoryl lipid A,liposomes, and derivatives and combinations thereof.
 9. The compositionof claim 4 wherein the composition further comprises one or moreadditional immunoregulatory agents.
 10. The composition of claim 9wherein the immunoregulatory agent is a substance selected from thegroup consisting of antigens of one or more strains of C. jejuni,antigens of ETEC, Shigella lipopolysaccharide structures, andunconjugated carrier proteins.
 11. The composition of claim 4 whereinthe subject is a human.
 12. A method of inducing an immune responseagainst C. jejuni in a subject comprising administering to the subjectan effective amount of the immunogenic synthetic construct of claim 1.13. The method of claim 12 wherein the subject is human.
 14. A method ofinducing an immune response against C. jejuni in a subject comprisingadministering to the subject an effective amount of the composition ofclaim
 4. 15. The method of claim 14 wherein the subject is human.
 16. Amethod of inducing an immune response against C. jejuni in a subjectcomprising administering to the subject an effective amount of thecomposition of claim
 5. 17. The method of claim 16 wherein said subjectis a human.
 18. A method of inducing an immune response against C.jejuni in a subject, said method comprising (a.) administering to thesubject an effective amount of the immunogenic synthetic construct ofclaim 1; and (b.) optionally administering to the subject one or moreboosting doses of the immunogenic synthetic construct administered instep (a).
 19. The method of claim 18 wherein the effective amountadministered in step (a) is from about 0.1 μg to about 10 mg of theimmunogenic synthetic construct.
 20. The method of claim 18 wherein saidmethod further comprises administering an adjuvant with the construct instep (a) and/or step (b).
 21. A method of inducing an immune responseagainst C. jejuni in a subject, said method comprising (a).administering to the subject an effective amount of the composition ofclaim 5; and (b). optionally administering to the subject one or moreboosting doses of the composition administered in step (a).
 22. Themethod of claim 21 wherein the effective amount administered in step (a)is from about 0.1 μg to about 10 mg of immunogenic synthetic construct.23. The method of claim 21 wherein said method further comprisesadministering an adjuvant with the construct in step (a) and/or step(b).
 24. The method of claim 16 wherein the composition is a vaccineformulation.
 25. The method of claim 21 wherein the composition is avaccine formulation.